Looking at Biomarkers
SMART (Strategies for Management of Antiretroviral Therapy) enrolled more than 5,000 HIV positive adults at more than 300 sites worldwide starting in 2000. Participants started the study with a CD4 count above 350 cells/mm3, and were randomly assigned either to stay on continuous ART or to suspend treatment when their CD4 cell count was above this level, resuming when it fell below 250 cells/mm3.
The treatment interruption arm was discontinued ahead of schedule in January 2006 after an interim analysis showed that participants in that group had higher rates of illness and death than those receiving continuous therapy.
While the increase in OIs and AIDS-related death was not surprising -- given that these people spent more time with lower CD4 counts -- the investigators also found that participants who periodically stopped treatment had a higher rate of serious non-AIDS conditions, including heart, liver, and kidney disease. This was unexpected, since the researchers had hypothesized that people who spent less time on therapy should have lower rates of complications attributed to antiretroviral drugs.
Presenting the data at the XVI International AIDS Conference the following summer, Jens Lundgren from the University of Copenhagen suggested there must be some "missing link" behind the excess risk of death among participants in the treatment interruption arm.
The SMART findings spurred an intensive search for an explanation, one that would usher in a new way of looking at HIV disease. The diversity of manifestations in this and other studies -- ranging from heart and liver disease to cancer and neurocognitive impairment -- suggests a common denominator underlying the "badness" resulting from chronic HIV infection even in the absence of advanced immune suppression.
This search led researchers to look at various biomarkers, or compounds in the blood that offer clues to inflammation, coagulation, and dysfunction of the endothelial cells lining the blood vessels (see table for a list of some commonly used biomarkers).
At the 2008 CROI, Lewis Kuller from the SMART team reported that elevated levels of the inflammation biomarker IL-6 and the coagulation marker D-dimer at study entry were strongly associated with increased cardiovascular and all-cause mortality; high-sensitivity CRP had a weaker correlation. IL-6 and D-dimer rose along with viral load after treatment interruption, while remaining stable in people on continuous therapy.
In a published report of these findings in PLoS Medicine, the investigators noted that people with the highest CRP values were twice as likely to die as those with the lowest levels. Those with the highest IL-6 levels had an eight-fold greater risk of death, while the highest D-dimer values were associated with a 12-fold increase. This pattern, the researchers wrote, "suggests that HIV infection results in activation of coagulation and inflammatory pathways that may impact multiple organs."
In follow-up reports, SMART investigators revealed that elevated biomarker levels in the treatment interruption group decreased but did not normalize after resuming therapy, and that the link between elevated baseline inflammation biomarkers and death persisted after more than two years of follow-up.
Another treatment interruption trial presented at CROI in 2008 also demonstrated a link between viral suppression and biomarkers of inflammation and endothelial dysfunction. Alexandra Calmy and colleagues measured blood biomarkers from 145 participants in STACCATO, the Swiss-Thai-Australia Treatment Interruption Trial. Compared with SMART, these participants had more advanced HIV disease, fewer traditional cardiovascular risk factors, and resumed therapy when their CD4 count fell below 350 rather than 250 cells/mm3.
After starting ART, levels of D-dimer, soluble vascular cell adhesion molecule 1 (VCAM-1), P-selectin, monocyte chemoattractant protein 1 (MCP-1), and leptin decreased as HIV was suppressed. Levels rose following treatment interruption, but did not fall back to baseline levels when therapy resumed. Conversely, anti-inflammatory biomarkers including IL-10 and adiponectin increased as viral load declined and fell during treatment breaks. In this cohort, however, there were no significant changes in IL-6 or CRP; because no participants experienced cardiovascular events or died, the researchers could not determine whether there was a link between biomarkers and clinical outcomes.
Another large trial looked at biomarker differences between people with and without HIV. Jacqueline Neuhaus and colleagues compared SMART participants versus HIV negative individuals in two large population-based cardiovascular studies, MESA (Multi-Ethnic Study of Atherosclerosis; 5,386 participants aged 45-76 years) and CARDIA (Coronary Artery Risk Development in Young Adults; 3,231 participants aged 33-44 years).
Overall, people with HIV had significantly higher levels of IL-6, high-sensitivity CRP, and D-dimer (all 50% to >100% higher), as well as cystatin C, a marker of kidney impairment. Biomarker levels were higher among SMART participants both on and off ART relative to the HIV negative groups, and the pattern held after adjusting for traditional risk factors.
In the years since the initial SMART report, more and more studies have measured biomarkers of inflammation, coagulation, and endothelial dysfunction. In the January 15, 2010, Journal of Infectious Diseases, for example, Jason Baker and colleagues reported that inflammation is apparent in HIV positive people with CD4 cell counts high enough that they do not yet require treatment. Untreated people with HIV had IL-6, D-dimer, and soluble intercellular adhesion molecule-1 (ICAM-1) levels 65%-70% higher than those of uninfected individuals.
Inflammation is also evident in people on stable ART with consistently suppressed viral load. Michael Boger and colleagues recently reported that HIV positive people with undetectable HIV RNA and low Framingham cardiovascular risk scores still had elevated CRP; half, in fact, had CRP levels greater than 3 mg/dL, suggesting a high risk of future cardiovascular events despite an absence of traditional risk factors.
Effects of Inflammation on the Body
In order to understand the implications of ongoing inflammation in people with HIV, it is useful to look at some specific conditions in more depth. Much of the research associating inflammation with disease has been carried out in HIV negative populations, but can offer insights for HIV positive people as well.
AIDS and CD4 Recovery
As noted above, research from early in the epidemic showed that immune activation markers were associated with progression to AIDS, and a similar link has been demonstrated for inflammation biomarkers such as CRP. But correlation does not prove that inflammation causes disease progression or vice versa.
Relatively few studies have assessed the connection between inflammation and OIs. But a SMART analysis presented by Alison Rodger and colleagues at the 2009 CROI showed that elevated CRP and IL-6 levels predicted development of opportunistic disease as well as non-AIDS conditions, independent of CD4 cell count and HIV viral load.
In contrast, there is an extensive body of research looking at the hallmark of HIV infection, CD4 T-cell depletion. In 2008, Hunt and the UCSF team presented two studies at CROI, the first showing that among untreated individuals with chronic HIV infection, increasing T-cell immune activation (indicated by CD38/HLA-DR expression) was strongly correlated with falling CD4 counts. The second, looking at elite controllers with naturally suppressed viral load, found that greater CD4 and CD8 T-cell activation was again strongly associated with lower CD4 counts -- even though the median remained close to 700 cells/mm3.
Studies have also consistently shown that T-cell activation increases the likelihood of poor CD4 cell recovery despite combination ART that fully suppresses plasma HIV replication, a phenomenon known as discordant response. Hunt and colleagues found this to be true both in their San Francisco cohort and in a group of patients starting ART in Uganda.
Chronic HIV infection is characterized by premature immunosenescence, or loss of immune cell function. T-cells and other leukocytes normally proliferate in response to pathogens, but cells have a built-in maximum number of divisions; once this limit is reached, they can no longer multiply in response to threats.
As Seema Desai and Alan Landay explained in the February 2010 issue of Current HIV/AIDS Reports, ongoing immune activation and inflammation due to constant stimulation by HIV or other chronic infections accelerates the process of T-cell exhaustion that occurs during the normal course of aging. T-cell homeostasis -- the balance between cell production and cell death -- may also be disrupted by changes in cytokine levels and premature CD4 and CD8 cell apoptosis.
Reduced T-cell regeneration is thought to be attributable in part to atrophy or shrinkage of the thymus; large numbers of new T-cells mature in the thymus from late fetal life through puberty, but the organ is typically mostly inactive in older adults. In addition, other lymphoid organs and tissues that can produce T-cells undergo fibrosis, or replacement of functional cells with scar material.
Middle-aged people with HIV show evidence of immunosenescence resembling that of HIV negative individuals two decades older. This includes low naive-to-memory cell ratio, reversal of the normal CD4-to-CD8 cell ratio, reduced T-cell proliferation, more cells with apoptosis markers, and more exhausted immune cells expressing CD57 without CD28. Once it occurs, T-cell senescence fails to normalize even with suppressive ART.
Accelerated Aging and Frailty
The immune system is not the only thing that ages prematurely in people with HIV. The types of chronic progressive conditions seen with greater frequency among HIV positive individuals -- heart disease, cancers, kidney disease, bone loss, cognitive impairment -- are the same ones that plague aging HIV negative people.
However, these conditions typically occur sooner in the setting of HIV disease, helping explain why HIV positive people as a group still do not achieve a normal lifespan. Various studies have shown, for example, that HIV positive people have blood vessel function resembling that of HIV negative counterparts ten or more years older, while neurological activity is similar to that of people 15-20 years older.
As researchers in medical fields ranging from cardiology to oncology have discovered, all of these conditions are linked to inflammation, leading Claudio Franceschi of the University of Bologna to coin the term "inflamm-aging."
Numerous studies over many years have shown that elderly people (usually defined as age 65-70 or older) have elevated levels of inflammation biomarkers compared with younger individuals; among women, a more pro-inflammatory pattern emerges after menopause.
Elderly people not only are more prone to specific organ diseases, they also can experience "frailty" characterized by unintentional weight loss, exhaustion, weakness, motor slowness, and a low level of physical activity. The MACS investigators reported that people with HIV were about 5-10 times more likely to develop frailty than uninfected individuals of the same age, and studies in the HIV negative population have linked frailty to inflammation.
Given that half of HIV positive people in the U.S. will be over age 50 by 2015, age-related complications and their causes will increasingly be a central aspect of HIV management (see "HIV and Aging," BETA, Summer/Fall 2009).
In the HIV negative general population, the role of inflammation has been most clearly demonstrated for cardiovascular disease. This link may be even stronger for people with HIV, who face the triple threat of increased inflammation, ART toxicities, and higher frequency of traditional risk factors.
Atherosclerosis ("hardening of the arteries") is a progressive process in which artery walls thicken and lose their elasticity as they fill up with plaques made up of accumulated lipids, immune cells, calcium, and scar tissue. Over time, this leads to impaired blood flow, which can ultimately result in a heart attack or stroke.
Over the past two decades, it has become increasingly clear that atherosclerosis is not just a matter of cholesterol passively building up in the arteries, but rather involves an active inflammatory process. This begins with damage to endothelial cells lining the blood vessels, which dilate and constrict the vessels to accommodate physiological demands. HIV proteins may directly contribute to endothelial dysfunction by altering cell-signaling pathways; CRP may also have a direct effect on endothelial function.
The earliest stage of plaque formation involves accumulation of "fatty streaks" of lipids including LDL cholesterol on the endothelial lining; this happens especially at artery forks or bifurcations where blood flow dynamics change. As lipids oxidize, they attract immune cells, primarily macrophages, which release pro-inflammatory signals. T-cells arrive, and in turn produce their own cytokines. The resulting inflammatory cascade releases "downstream" molecules such as CRP that can be measured in circulating blood.
The inflamed endothelial lining around these lesions expresses adhesion molecules -- including VCAM-1, ICAM-1, and selectins -- that enable leukocytes to move along and bind to the endothelial lining. Lipids and immune cells eventually breach this lining and build up in the intima, or innermost layer of the artery wall.
There, scavenger macrophages continue to ingest oxidized LDL, becoming foam cells. Signals released by activated immune cells promote fibrogenesis, or production of extracellular structural material such as collagen that forms a fibrous cap over the lipid core of a plaque; around the outer edges, plaques accumulate calcium and harden.
But other inflammatory chemicals act to weaken the cap, causing it to rupture. This triggers formation of blood clots (assisted by fibrinogen), furthering narrowing the artery. Clots are broken down by plasminogen, releasing D-dimer as a byproduct. Pieces of plaques or clots can break away and become lodged in smaller vessels, causing complete blockage.
As plaques build up in the coronary arteries supplying the heart muscle, they deliver less oxygen (known as ischemia), which can cause angina, or chest pains. Complete blockage of the coronary arteries leads to myocardial infarction, or heart attack. Blockage of the carotid arteries supplying the brain leads to strokes.
Inflammation helps explain the effects of traditional cardiovascular risk factors. Cigarette smoke, for example, is a toxin that promotes inflammation. Excess body fat produces pro-inflammatory compounds, as discussed above. LDL cholesterol builds up in artery walls and triggers inflammatory responses, while HDL cholesterol removes LDL from the arteries and has anti-inflammatory effects.
Several large general population studies have shown that circulating blood levels of compounds involved in atherosclerosis -- including IL-6, VCAM-1, selectins, fibrinogen, D-dimer, and especially CRP -- can predict future cardiovascular events. Not coincidentally, these are the same markers linked to cardiovascular events and death in SMART and other studies of people with HIV.
People with chronic HIV infection -- even those on suppressive ART -- are more likely than HIV negative individuals to have signs of endothelial dysfunction and subclinical atherosclerosis. These include decreased flow-mediated dilation (how well arteries respond to changes in blood flow), reduced arterial distensibility (ability to dilate), increased arterial stiffness, and greater intima-media thickness (IMT), or width of the artery wall.
At this year's CROI, Hsue's group reported that HIV positive people in the San Francisco SCOPE cohort experienced more rapid atherosclerosis progression than HIV negative individuals over two years, as determined by measuring IMT at the bifurcation of the carotid artery. This was seen in people with undetectable viral load on ART and even elite controllers. IMT progression was associated with inflammation as indicated by elevated CRP levels.
When the investigators looked at flow-mediated dilation of the brachial (upper arm) artery, they again found that HIV positive people had significantly worse measurements than uninfected people. Elevated CRP was a stronger predictor of impaired dilation than older age in the HIV positive group, while the opposite was true in the HIV negative group.
Robert Kaplan and colleagues reported at CROI that increased carotid artery IMT and reduced distensibility correlated with greater CD4 and CD8 cell activation and T-cell senescence among HIV positive participants (but not among HIV negative women) in the Women's Interagency HIV Study.
What about clinical events? In the July 2009 Journal of Acquired Immune Deficiency Syndromes, Virginia Triant and colleagues reported that in a retrospective analysis of patients seen at Massachusetts General and Brigham and Women's Hospitals in Boston, HIV infection and elevated CRP both more than doubled the risk of acute myocardial infarction. These factors had an additive effect, so HIV positive people with high CRP had a four-fold higher risk relative to HIV negative people with normal CRP.
Summing up her research, Hsue said these studies support the "common theme that independent of drug therapy, independent of viremia and viral load, there is some kind of badness associated with HIV infection in terms of cardiovascular risk."
Liver disease has become a major cause of illness and death in people with HIV in the combination ART era. This is often due to hepatitis B or C coinfection, but drug toxicity may also play a role. Viral hepatitis, drug toxicity, heavy alcohol use, and other causes of injury promote liver inflammation signaled by elevated levels of alanine transaminase (ALT) and other liver enzymes.
Over time, chronic liver inflammation can lead to fibrosis and cirrhosis, as the liver attempts to heal itself by producing extracellular scar material. The associated altered chemical signaling and oxidative stress can promote development of hepatocellular carcinoma, a form of liver cancer.
The impact of systemic immune activation and persistently elevated pro-inflammatory cytokines on liver disease is not fully understood. TNF-alpha and IL-6, for example, appear to have paradoxical effects on the liver, promoting some types of liver injury while protecting against others.
Like HIV, HBV and HCV can directly trigger immune activation and alter cytokine signaling. Stepped-up immune activation from fighting two viruses at once may help explain the more rapid liver fibrosis progression -- and possibly worse HIV disease progression -- seen among HIV/HCV and HIV/HBV coinfected people.
Liver disease was one of the non-AIDS conditions that increased among participants undergoing treatment interruption in the SMART trial. HIV/HBV or HIV/HCV coinfected people had a higher rate of death due to non-opportunistic causes even after controlling for liver cancer.
At this year's CROI, Lars Peters and colleagues showed that elevated hyaluronic acid (a component of extra-cellular material used as a biomarker for liver fibrosis) was associated with higher levels of CRP, IL-6, and D-dimer in HIV/HBV and HIV/HCV coinfected SMART participants, predicting a four- to six-fold increase in the risk of non-AIDS death. They suggested that coinfected patients with impaired liver function are "particularly in a pro-inflammatory state" that might be exacerbated by ART interruption.
People with HIV are susceptible to HIV-associated nephropathy (a form of chronic kidney failure) as well as kidney toxicity due to certain antiretroviral drugs (see "Renal Complications of HIV/AIDS," BETA, Summer 2007). Kidney disease was also among the non-AIDS conditions that increased during ART interruption in SMART.
Kidney disease plays a role in the nexus of metabolic abnormalities linked to inflammation. Studies have shown that compounds involved in inflammation, coagulation, and endothelial dysfunction (including CRP, cell adhesion molecules, MCP-1, and fibrinogen) are elevated in people with kidney impairment.
As Dena Rifkin and Michael Sarnak explained in the April 2009 American Journal of Kidney Disease, glomerulosclerosis -- the hardening of capillary webs that filter blood in the kidneys -- in many ways resembles atherosclerosis, featuring accumulation of cholesterol, migration of monocytes, production of lipid-laden macrophages, and fibrosis, suggesting that the same inflammatory processes are likely involved.
Despite widespread ART use, people with HIV continue to develop a spectrum of cognitive, motor, and psychological manifestations ranging from asymptomatic neurocognitive impairment to HIV dementia (see "HIV and the Brain," BETA, Summer/Fall 2009). As with other non-AIDS conditions, neurocognitive impairment improves with ART, but does not completely normalize.
HIV enters the brain soon after initial infection, hiding in macrophages to cross the protective blood-brain barrier. Within the central nervous system (CNS), the virus does not infect neurons (the cells that transmit electrical impulses responsible for thought and movement), but mainly targets specialized brain macrophages called microglia. Once inside these long-lived cells, it can remain latent for an extended period.
HIV typically does not directly kill brain cells in large numbers, but rather sets off a cascade of damaging inflammatory changes; in addition, HIV Tat and gp120 proteins have direct neurotoxic effects. Immune cells activated to fight the virus produce pro-inflammatory cytokines such as TNF-alpha and interferons, which activate astrocytes (brain support cells) and attract more leukocytes. This inflammatory cascade disrupts cellular communication channels and leads to oxidative stress.
The extent of the inflammatory response in the brain does not appear to closely correspond to the amount of HIV present; even residual virus in people on suppressive ART may be enough to sustain a self-perpetuating inflammatory response. In addition to the direct effects of chemicals in the brain, broader metabolic abnormalities and vascular changes linked to inflammation also contribute to cognitive impairment.
It is increasingly clear that these factors also play a role in Alzheimer's disease and other types of age-related cognitive decline. This suggests that as HIV positive people age, they may be prone to additive neurocognitive impairment due to multiple processes.
Several large studies indicate that people with HIV have higher rates of cancers not traditionally considered AIDS-related. Some, including anal cancer and liver cancer, have known infectious causes, but others do not. While cancer risk increases with lower CD4 counts, there appears to be excess risk even among people with well-preserved immune function.
Outside the HIV field there has been extensive research looking at the link between immune activation, inflammation, and cancer. In June 2004, the U.S. National Cancer Institute (NCI) held a meeting on inflammation and cancer, one of a series of cancer biology think tanks.
"It is generally accepted that chronic inflammation -- triggered by toxins, microbes, or autoimmune reactions -- plays a major role as a tumor promoter," the participating experts summarized.
The immune system fights disease that originates within the body as well as invasions coming from outside. CD8 T-cells and natural killer cells maintain surveillance of cells throughout the body and destroy those that show signs of abnormal growth, or malignant transformation. But the signaling cascades that direct the normal processes of cell growth and tissue repair can go awry, resulting in uncontrolled cell proliferation and invasion of healthy tissue.
Initially, inflammation contributes to cancer development by causing oxidative stress and DNA damage. Over time, chronic inflammation appears to promote cancer cell survival and proliferation. In another vicious cycle, tumor cells produce compounds that attract immune cells, which in turn secrete chemicals that stimulate further cancer proliferation. In addition, cancer cells can co-opt chemokines, cell adhesion molecules, and other inflammatory mediators (intermediate chemicals) to facilitate their migration.
Several large general population studies have linked a variety of cancers to elevated levels of inflammation markers, including CRP. Since the SMART trial, investigators have started to search for these associations in people with HIV, and evidence to date suggests a similar link.
Finally, chronic inflammation has been shown to have an effect on mortality itself, as evidenced by studies presented at this year's CROI. Hunt's team reported that in their study of people starting ART in Uganda, patients with greater CD8 T-cell activation before starting treatment had shorter survival times than those with less activation, despite good HIV viral load suppression.
Phyllis Tien and fellow investigators looked at the relationship between biomarkers and mortality among more than 900 participants in the FRAM Study (Fat Redistribution and Metabolic Change in HIV Infection) over five years. After adjusting for HIV status and traditional cardiovascular risk factors, baseline levels of both CRP and fibrinogen were strongly associated with death during follow-up. "Our findings suggest that even in those with apparent restoration of CD4 cells, inflammation remains an important risk factor for mortality," they concluded.
Investigators with the INSIGHT collaboration looked for a similar association among participants in the FIRST study, which compared three types of first-line antiretroviral regimens in people with advanced HIV/AIDS. Participants who progressed to AIDS, immune reconstitution inflammatory syndrome (IRIS), or death within 12 months of starting ART had higher baseline biomarker levels. "Elevated levels of biomarkers associated with inflammation (CRP, IL-6), coagulation (D-dimer), and tissue fibrosis (hyaluronic acid) in ART-naive patients who have good virologic response may be useful in identifying patients at risk for AIDS or death during the first year of ART," the researchers concluded.