This study shows for the first time that a mixture of two compounds derived from silymarin and formulated for intravenous dosing inhibited HIV-1 infection in multiple cell types including human PBMCs, CEM cells, and TZM-bl cells. SIL's anti-HIV effects were seen against four viruses. The antiviral effect of SIL in T cells was associated with a reduction of stimulus-induced (PHA, IL-2) expansion of PBMC and primary CD4+ T cell cultures, and a reduction in CEM cell expansion in the absence of stimulation. Thus, SIL caused a generalized attenuation of T cell functions required for proliferation, activation potential and/or status, and HIV infection. The fact that SIL inhibited the expansion of both T and B cell subsets () and reduced the number but not proportions of CD4+ T cells expressing CXCR4 and/or CCR5, suggests that effects of SIL are generalized and not confined to a particular T cell subset. Therefore, as for HCV infection, SIL may modulate fundamental metabolic processes that result in the blunting of multiple cellular responses including activation and proliferation, which ultimately renders CD4+ T cells less susceptible to HIV-1 infection.
SIL suppression of T cell expansion did not involve cell death via cell cycle arrest, apoptosis or necrosis, nor inhibition of T cell subsets within PBMC cultures. This observation extends previous findings that in the context of HCV infection, silymarin, SIL, and silymarin-derived pure compounds inhibit T cell growth 
. While SIL inhibited both primary and tumor T cells to similar degrees, SIL did not seem to inhibit the growth of adherent cells such as TZM-bl cells (as shown in this study) and human hepatoma Huh7 cells 
. Additional studies are required to determine if SIL causes subtle growth and/or metabolic responses in adherent HIV-1 target cells, such as macrophages.
The current report focused on in vitro infection of PBMCs from HIV seronegative subjects. We speculate that since silymarin and silymarin-derived compounds and mixtures appears to block T cell activation by many stimuli including IL-2 (this study), PHA 
, and by anti-CD3 stimulation 
, we predict that SIL will also block stimulus-induced activation of virus replication in chronically infected cells.
Cumulatively, the data presented in this report suggest that SIL inhibited viruses by multiple inhibitory mechanisms that target the cell and reduce the activation status/potential. However, it is possible that SIL could hit virus-specific targets, because SIL and other silymarin-derived compounds are weak inhibitors of HCV NS5B polymerase activity in vitro 
. At present, we can only speculate which point(s) in the HIV lifecycle are impaired by SIL. This is particularly important because we showed that SIL inhibited a CXCR4 using virus (LAI) more effectively than CCR5-using viruses (BAL, and two pseudoviruses), yet SIL did not cause a preferential down-regulation of CXCR4 over CCR5 co-receptors. However, since we only tested a single CXCR4-using virus in the present study, some caution in interpreting these results is warranted. While SIL inhibits HCV entry by blocking fusion of viral membranes with endosomal membranes as well as transmission of progeny viruses 
, HIV enters cells by fusion of viral and plasma membranes 
. However, we have recently found that silibinin (the parent mixture of SIL) and SIL inhibits clathrin-mediated endocytosis and thereby inhibits viruses like HCV, vesicular stomatitis virus, reovirus, and influenza virus, which exploit the clathrin pathway to gain entry into cells (manuscript in preparation). Since HIV may also use clathrin to enter and traffic within cells 
, SIL could inhibit HIV entry, possibly by blocking clathrin. Additional studies will be required to define where SIL acts on the HIV lifecycle and what accounts for the apparent differential suppression of CXCR4- versus CCR5-using viruses.
It has been previously shown that in both humans and in non-human primates, HIV-1 causes immune depletion at submucosal sites, such as the gastrointestinal track, followed by the onset of generalized immune activation 
, manifested as increased frequencies of T cells expressing activation markers, increased expression of pro-inflammatory cytokines, continuous immune cell turnover, culminating in T and B lymphocyte exhaustion. Since SIL reduced expression of activation markers on CD4+ T cells (), the immunomodulatory effects of SIL may be clinically relevant for reducing HIV-induced immune activation. In addition, because SIL blocks HCV re-infection during liver transplantation 
as well as HCV entry into hepatocytes 
, silymarin-derived compounds and mixtures might have utility as a component of topical microbicide therapy 
to prevent HIV infection, provided these compounds can be shown to inhibit HIV entry. Further investigation of the effects of silymarin-derived compounds in immune activation and transmission of HIV at mucosal sites is warranted.
In patients with HIV/HCV co-infections, T-cell activation can be higher than in patients infected with HIV alone 
. Because silymarin and SIL suppress T cell proliferation 
, silymarin-derived compounds may also be useful in controlling immune activation in co-infected patients. Moreover, silymarin-derived compounds and mixtures such as SIL may help reduce both HIV- and HCV-induced oxidative stress. For example, HCV infection induces oxidative stress and inflammation 
, and silymarin-derived compounds block HCV-induced oxidative stress directly by inhibiting virus replication as well as independently of suppression of viremia 
. For HIV, there are several examples of gp120-mediated neurotoxicity involving oxidative stress, as well as numerous studies supporting the concept that oxidative stress is involved in the progression of HIV disease 
. Therefore, silymarin-derived compounds may represent novel components of a multi-pronged approach to eradicate HCV, suppress HIV replication and transmission, and reduce immune activation and inflammatory disease in subjects with chronic HIV and/or HCV infection.