Eradication of latent HIV-1 reservoirs, which are routinely studied in circulating T cells, is a major clinical challenge. However, increasing evidence suggests the existence of other cellular HIV-1 reservoirs, including tissue-resident macrophages. A new study now reports that urethral macrophages are a principal reservoir of replication-competent HIV-1 in penile tissues from HIV-1-infected individuals undergoing suppressive combination antiretroviral therapy (cART).

Credit: Pat Morgan/Springer Nature Limited

“We previously discovered that, upon entry into the penile urethra, HIV-1 targets urethral macrophages, but not urethral T cells, which led us to investigate whether urethral tissue macrophages could form replication-competent HIV-1 reservoirs,” explains lead investigator Yonatan Ganor. Accordingly, the authors evaluated whole penile tissues — recovered during elective gender reassignment surgery — from HIV-1-infected individuals undergoing cART who had undetectable plasma viral loads (HIV-1–cART individuals; n = 20).

The authors first used nested PCR and in situ hybridization to investigate the presence of integrated HIV-1 DNA in urethral tissues from HIV-1–cART individuals and uninfected controls. Strikingly, HIV-1-specific products were detected in HIV-1–cART, but not control, tissues. Furthermore, integrated HIV-1 DNA was only detected in CD68+ macrophages, but was undetectable in CD3+ T cells, suggesting that urethral macrophages are the main population containing integrated HIV-1 DNA. In addition, HIV-1 RNA was detected in CD68+ macrophages but not CD3+ T cells from HIV-1–cART urethral tissues, showing that HIV-1 RNA persists in urethral macrophages during suppressive cART.

The investigators also developed a viral outgrowth assay adapted to tissue macrophages that provided a readout of the outgrowth of replication-competent infectious HIV-1 in single-cell suspensions prepared from the epithelial and stromal compartments of HIV-1–cART urethral tissues. Treatment with lipopolysaccharide (LPS), a latency-reverting agent (LRA) that specifically reactivates HIV-1 production in macrophages, induced HIV-1 outgrowth. However, HIV-1 outgrowth was not detected following treatment with the T cell-specific LRA phytohaemagglutinin, suggesting that macrophages, but not T cells, form an HIV-1 reservoir in these tissues. In confirmation, depletion of macrophages using magnetic beads in combined epithelial–stromal cell suspensions almost completely abolished HIV-1 outgrowth on LPS treatment.

In line with DNA and RNA level data, fluorescence microscopy revealed that the HIV-1 capsid protein p24 colocalized with CD4+CD68+ macrophages in the urethral stroma of HIV-1–cART individuals and that p24 was negative in stromal CD68–CD4+ T cells. Further morphological assessment of HIV-1-containing compartments in macrophages using confocal microscopy showed that HIV-1 proteins were only detected within CD68+ macrophages (and not CD3+ T cells) and were contained only within intracellular structures, inferring HIV-1 compartmentalization within virus-containing compartments (VCCs). Accordingly, using electron microscopy, epithelial and stromal urethral macrophages were shown to harbour intracellular VCC-like vesicular structures that enclosed HIV-1 virions. Finally, multiparametric flow cytometry coupled with immunofluorescence microscopy experiments demonstrated that a unique subset of macrophages with intermediate polarization (termed Mi) — which co-express M1 and M2 polarization markers — were enriched in urethral tissues from HIV-1–cART individuals and preferentially contain HIV-1.

In summary, the study reveals that macrophages are a major HIV-1 reservoir in the penile urethra of HIV-1–cART individuals, challenging the dogma that HIV-1 reservoirs principally reside in T cells.

macrophages are a major HIV-1 reservoir in the penile urethra

The findings also have clear relevance for therapeutic HIV-1 eradication strategies “This information is crucial for ‘shock and kill’ strategies aimed at reservoir elimination. To be effective, such new strategies should also consider, and be adapted to, purging HIV-1 from reservoirs in macrophages,” adds Ganor. “For instance, we are investigating whether LPS–TLR4 signalling and clinically approved TLR4 agonists could be exploited to reactivate this newly identified macrophage reservoir, in order to achieve the clinical goal of HIV-1 eradication.”