CARD8 inflammasome activation during HIV-1 cell-to-cell transmission

Joint Public Review:

Following up on their previous work, the authors investigated whether cell-to-cell transmission of HIV-1 activates the CARD8 inflammasome in macrophages, an important question given that inflammasome activation in myeloid cells triggers proinflammatory cytokine release. The data support the idea that CARD8 is activated by the viral protease and promotes inflammation. However, time-course analyses in primary T cells and macrophages and further information on the specific inflammasome involved would further increase the significance of the study.

Strengths:

The manuscript is well-written and the data is of good quality. The evidence that CARD8 senses the HIV-1 protease in the context of cell-to-cell transmission is important since cell-to-cell transmission is thought to play a key role in viral spread in vivo, and inflammation is a major driver of disease progression. Clean knockout experiments in primary macrophages are a notable strength and the results clearly support the role of CARD8 in protease-dependent sensing of viral spread and the induction of IL1β release and cell death. The finding that HIV-1 strains are resistant to protease inhibitors differ in CARD8 activation and IL1β production is interesting and underscores the potential clinical relevance of these results.

Weaknesses:

One weakness is that the authors used T cell lines which might not faithfully reflect the efficiency of HIV-1 production and cell-cell transfer by primary T cells. To assess whether CARD8 is also activated by protease from incoming viral particles earlier time points should be analyzed. Finally, while the authors exclude the role of NLRP3 in IL-1b and the death of macrophages it would be interesting to know whether the effect is still Gasdermin D dependent.

Author response:

Thank you for the positive and constructive feedback on our manuscript. We appreciate you highlighting the importance of our work advancing our understanding of the molecular etiology of acquired immunodeficiency syndrome (AIDS). To extend and further substantiate the observation that the CARD8 inflammasome is activated in response to viral protease during HIV-1 cell-to-cell transmission, we are in the process of completing additional experiments that are responsive to reviewer feedback, including:

• Primary CD4+ T cell to monocyte-derived macrophage (MDM) transmission: We have now repeated the cell-to-cell experiments with HIV-1 transfer from primary CD4+ T cells to primary monocyte-derived macrophages, and our findings are consistent with CARD8-dependent IL-1β release from HIV-1-infected macrophages in this more physiologic context. We are in the process of repeating these experiments with additional donors and will add these results to the revised manuscript.

• Heterogeneity amongst blood donors: We have now repeated the cell-to-cell transfer and CARD8 knockout in MDMs with additional donors. While we continue to observe heterogeneity amongst donors, the key observation that CARD8 is require for inflammasome responses to HIV-1 infection is consistent. We note that some donors, including the one individual reported in the first submission, have markedly diminished CARD8 activity (to both HIV-1 and VbP).

• Time course experiments: We did conduct a time course experiment when initially establishing these assays. We have now repeated these experiments with additional timepoints and in the presence or absence of the RT inhibitor nevirapine. The results of these experiments will be included in the revised manuscript.

• The role of Gasdermin D: We are mostly interested in the release of IL-1β from the infected macrophages due to its potential contribution to myeloid-driven inflammation in PLWH. To date, there is no evidence that any other pore-forming protein other than GSDMD can initiate IL-1β release (and pyroptosis) downstream of CARD8. Nonetheless, we will attempt this experiment with the Gasdermin D inhibitor, disulfiram.

We believe these and other experiments will further support the importance of the CARD8 inflammasome in myeloid-driven inflammation in PLWH and look forward to submitting the revision.