The TLR7/IRF-5 Axis in HIV: a Matter of Life and Cell Death?

Human immunodeficiency virus (HIV) is a retrovirus that manifests in the progressive loss of CD4 T cells, subsequently leading to a vulnerability to opportunistic infections in people living with HIV (PLWH) (Waymack and Sundareshan 2023). The WHO reported that in 2022 there were approximately 39 million PLWH worldwide and 630,000 people who had died as a result of HIV-related illnesses.

HIV primarily infects and replicates within activated CD4 T cells, causing their death via mechanisms such as CD8 T cell-mediated killing, pyroptosis in infected cells, and apoptosis in uninfected bystander cells. The typical half-life of HIV-infected CD4 T cells is around 12 to 36 hours.

Even with antiretroviral therapy (ART), which effectively controls replication of HIV, most PLWH have lingering low levels of inflammation and immune activation, as well as increased sensitivity to cell death, particularly in the memory CD4 T cell subset. However, the mechanisms behind this susceptibility to cell death have been largely unknown (Alimonte et al. 2003, Carmona-Pérez et al. 2023). In this blog, we discuss a recent study by Carmona-Pérez et al., in which they investigate the role of the Toll-like receptor (TLR) 7/interferon regulatory factor 5 (IRF-5) axis in the predisposition of CD4 T cells to apoptosis during HIV infection.

A Trip Down Memory Lane

The story starts with a previous study from Fabié et al., which found that IRF-5 (a transcription factor involved in activating type 1 interferon-associated and pro-inflammatory cytokine genes) was progressively upregulated in CD4 T cells, specifically effector and memory subsets, during chronic infection with Leishmania donovani. By adoptive transfer of CD4 T cells lacking TLR7, a known inducer of IRF-5, they observed that expression of IRF-5 could no longer be increased in these cells, indicating that TLR7 stimulation was responsible for this effect.

The researchers also noticed that, while wildtype (WT) CD4 T cells showed increasing levels of the apoptotic marker annexin V over the course of the infection, CD4 T cells lacking TLR7 were protected from the peak increase in annexin V typically observed by day 28, indicating their protection from cell death at this time point. To further pin down the mechanism of cell death, they analyzed receptors associated with triggering apoptosis, namely the Fas receptor (Fas) and tumor necrosis factor receptor-1 (TNFR-1). They found that expression levels of both Fas and TNFR-1 were unchanged between WT and TLR7 deficient CD4 T cells throughout the course of the infection, suggesting that neither Fas nor TNFR-1 was the target of TLR7 signaling in this model. The researchers also found that another key receptor that mediates apoptosis, death receptor 5 (DR5), was significantly upregulated in WT CD4 T cells from day 21 post infection onwards. This is around the same time that signs of apoptosis began to appear in the CD4 T cells.  This led them to hypothesize that during chronic infection, damage-associated molecular patterns (DAMPs), which are released following tissue damage caused by inflammation, trigger the stimulation of the TLR7/IRF-5/DR5 pathway, causing the death of CD4 T cells (Fabié et al. 2018).

Since HIV infection also involves a chronic inflammatory environment, an increased sensitivity to cell death, and has previously been shown to stimulate the upregulation of TLR7 in CD4 T cells, Carmona-Pérez et al. decided to investigate the contribution of the TLR7/IRF-5 pathway to memory CD4 T cell death in HIV.

Memory Loss

Even when PLWH receive ART and have the HIV virus under control, they still often have increased cell death in the memory CD4 T cell compartment. Under normal circumstances, memory cells facilitate a rapid recall response to a secondary challenge of a previously encountered pathogen. Therefore, a deficiency in these cells can cause major problems in individuals affected by chronic infection (Angelosanto et al. 2012). This begs the question is IRF-5 also responsible for the increased sensitivity of CD4 memory cells to cell death?

Carmona-Pérez et al. addressed this question by recruiting PLWH who were currently on ART and displayed viral suppression and CD4 T cell recovery of over 400 cells per μl of blood for at least five years (for ease, let’s call them the ART group), alongside so-called “elite controllers” (ECs), HIV patients who had undetectable levels of virus in the blood and CD4 T cell recovery without treatment for over five years. ECs are able to maintain CD4 T cell numbers in the blood without the need for ART, so it was of interest that IRF-5 frequencies were significantly lower in memory CD4 T cells in the EC group compared to the ART group. Furthermore, while annexin V expression correlated with IRF-5 expression in memory CD4 T cells from the ART group, no correlation was observed between these markers in EC memory cells.  

Could IRF-5 Inhibition Refresh Your Memory?

These thought-provoking findings raised two very important follow up questions. The first of which is what drives the upregulation of IRF-5 in these cells? Based on the group’s previous study, TLR7 seemed like a good place to start their investigation. Therefore, they analyzed the expression of TLR7 mRNA in CD4 T cells from the different groups and found that it was highest in the ART cohort, with intermediate levels in ECs compared to low levels in uninfected individuals. They then stimulated CD4 memory T cells from the different groups with a TLR7 agonist, IMQ, and observed the highest induction of IRF-5 expression in the ART group, followed again by a lesser increase in EC cells, and no significant difference in the HIV free individuals, when compared to their respective untreated controls.

Additionally, a significant correlation was found between IRF-5 expression and the amount of a DAMP associated with damage to the intestinal muscosa, regenerating islet-derived protein 3 α (REG3α), in the serum of ART patients, suggesting that IRF-5 may be triggered by TLR7 stimulation by DAMPs from the impaired intestinal mucosa.

The second question that arose was how does IRF-5 trigger apoptosis? Interestingly, unlike their previous study, DR5 expression did not differ between HIV-infected and uninfected individuals, and there was no correlation observed between DR5 and annexin V levels. On the other hand, mRNA levels of caspase 8, a molecule known to initiate apoptosis, were significantly increased in CD4 T cells from the ART group in comparison to the EC and uninfected groups, and this was further increased upon IMQ treatment in ART individuals but not in the HIV free group. By pretreating CD4 T cells from the different donor groups with IMQ, followed by recombinant Fas ligand (rFasL), the ligand for the apoptosis receptor Fas, the researchers noticed significantly higher dead cells in the ART donors compared with HIV free donors, indicating a predisposition toward Fas-mediated cell death in the treated individuals. The frequency of dead cells was notably lower upon treatment with IRF5 inhibitory peptides.

Overall, these results indicate that TLR7 stimulation caused by DAMPs released following cell death leads to IRF-5 expression and subsequent induction of the Fas/FasL and caspase 8 mediated cell death pathway in memory CD4 T cells from ART individuals. This induction of apoptosis will then, in turn, release more DAMPs and trigger further IRF-5 expression, in what the researchers aptly refer to as “a spiral of death”. This study also raises the possibility of IRF-5 blockade as a potential therapy against the loss of memory CD4 T cells not only in HIV patients receiving ART, but also potentially in individuals suffering from chronic viral infections.