Secreted factors induced by PKC modulators do not indirectly cause HIV latency reversal

Abstract

HIV can establish a long-lived latent infection in cells harboring integrated non-expressing proviruses. Latency reversing agents (LRAs), including protein kinase C (PKC) modulators, can induce expression of latent HIV, thereby reducing the latent reservoir in animal models. However, PKC modulators such as bryostatin-1 also cause cytokine upregulation in peripheral blood mononuclear cells (PBMCs), including cytokines that might independently reverse HIV latency. To determine whether cytokines induced by PKC modulators contribute to latency reversal, primary human PBMCs were treated with bryostatin-1 or the bryostatin analog SUW133, a superior LRA, and supernatant was collected. As anticipated, LRA-treated cell supernatant contained increased levels of cytokines compared to untreated cell supernatant. However, exposure of latently-infected cells with this supernatant did not result in latency reactivation. These results indicate that PKC modulators do not have significant indirect effects on HIV latency reversal in vitro and thus are targeted in their latency reversing ability.

Keywords: Bryostatin-1; HIV; Kick-and-kill approach; Latency reversal; PKC modulators; SUW133.

Fig. 1.. Schematic representation of study.

Primary human PBMC were isolated from 8 healthy human donors and combined with a latency reversing agent (LRA) in culture. Cells were washed after 24 h and incubated for a further 24 h then the supernatant was filtered and analyzed for cytokine composition (Fig. 2) and capacity to induce HIV latency reversal (Fig. 3).

Fig. 2.. Cytokines induced in primary human PBMC by PKC modulators.

Conditioned media (CM) generated with peripheral blood mononuclear cells (PBMC) from 8 (control and bryostatin-1) or 6 (SUW133) different healthy human donors were analyzed using a human cytokine 38-plex Luminex immunoassay. A) A heat map showing results of all 38 human cytokines is provided and the mean concentration values in pg/mL along with the standard error of the mean (SEM). The mean percentage change relative to media only control are also shown. Dark green indicates a significant increase (P < 0.05); light green indicating a non-significant increase; light red indicating a non-significant decrease; and dark red indicating a significant decrease (Mann-Whitney U test, p < 0.05). B) Example cytokine profiles from data shown in part A, with each color and shape representing results using PBMC from a different human donor.

Fig. 3.. HIV Latency Reversal in select J-Lat clones stimulated with conditioned media (CM).

A) Various Jurkat-Latency (J-Lat) clones were treated with direct LRA or conditioned media (CM) for 48 h. All control conditions (media only and direct stimulations) were done in technical duplicates, in three independent biological replicates, resulting in an n = 6; For conditioned media samples, technical duplicates in three independent biological replicates per donor (4 donors each) resulted in n = 24. B) Promonocytic U1 cells were treated with direct LRA or CM for 48 h. All control conditions were done in technical triplicate, in two independent biological replicates, resulting in an n = 6; For conditioned media samples, technical duplicates in two independent biological replicates per donor (4 donors each) resulted in n = 8. A two-tailed, unpaired, unequal variance Student’s t-Test was performed, with (*) indicating p < 0.05; and (**) indicating p < 0.001. Each color and shape correspond to PBMC from a different human donor.