The human immunodeficiency virus (HIV) affects millions of people across the world, and has caused over forty million deaths. HIV attacks the immune system, eventually leading to lower levels of immune cells, which prevent the body from fighting infections. One of the early effects of HIV infection is inflammation, an immune process that helps the body remove foreign invaders like viruses. Unfortunately, long term inflammation can lead to serious conditions like cardiovascular disease and cancer.

Doctors manage HIV using a class of drugs known as antiretrovirals. These drugs reduce the amount of virus in the body, but they cannot eliminate it entirely. This is because, in the early days of infection, copies of the virus build up in certain organs and tissues, like the gut, forming viral reservoirs. Antiretroviral drugs cannot reach these reservoirs to eliminate them, making a cure for HIV out of reach. One way to address this problem is to develop a new class of drugs that can stop the virus from forming these reservoirs in the first place.

Amand et al. wanted to see whether they could reduce the amount of viral reservoirs that form in HIV patients by interrupting a process called inflammasome activation, which occurs early after HIV infection. Inflammasomes are viral detectors that play a role in both inflammation and the formation of viral reservoirs. They activate an enzyme called caspase-1, which in turn activates proteins called cytokines. These cytokines go on to stimulate further inflammation.

Amand et al. wanted to see whether a drug called VX-765, which blocks the activity of the caspase-1 enzyme, could reduce inflammation and stop the formation of viral reservoirs. To do this, Amand et al. first ‘humanized’ mice, by populating them with human immune cells, so they could become infected with HIV. They then infected these mice with HIV, and proceeded to treat them with VX-765 two days after infection. The results showed that these mice had fewer viral reservoirs, lower levels of cytokines and higher numbers of immune cells than untreated mice.

The findings of Amand et al. show that targeting inflammasome activation early after infection could be a promising strategy for treating HIV. Indeed, if similar results were obtained in humans, then this technique may be the road towards a cure for this virus. In any case, it is likely that combining drugs like VX765 with antiretrovirals will improve long term outcomes for people with HIV.

Around 38 million people worldwide are living with the human immunodeficiency virus (HIV) and every year, 1. 7million people get newly infected (UNALDS, 2020). Although combined antiretroviral therapy (cART) can suppress viremia and dramatically improved the life expectancy and the clinical outcome of HIV-1 infected patients (Antiretroviral Therapy Cohort Collaboration, 2008), optimal cART treatment is not curative due to the persistence of a lifelong HIV-1 replication competent reservoir (Siliciano et al., 2003). In addition, cART does not fully resolve HIV-associated immune abnormalities including low-grade chronic inflammation, immune activation/dysfunction, and poor CD4⁺ T cell reconstitution (French et al., 2009; Hunt et al., 2003) and imposes a high burden to the health care system due to treatment resistance, adverse events, and high treatment costs. Recent evidence indicates that inflammation drives HIV-1 reservoirs persistence which in turn contributes to inflammation, creating a pathogenic vicious cycle (Klatt et al., 2013). These inflammatory and immune disorders predispose HIV-1 infected patients under cART to non-AIDS age-related comorbid conditions such as cardiovascular diseases, cancer, and neurodegenerative diseases and are predictive of an increased risk of morbidity and mortality (Kuller et al., 2008). Strategies targeting the mechanisms of HIV-induced inflammation are now evaluated to reduce HIV-1 reservoirs, a fundamental requirement to achieve an HIV cure (Massanella et al., 2016).

HIV infection triggers immune activation/dysfunction and inflammation through several mechanisms such as direct effects of viral proteins on immune cells (Rieckmann et al., 1991; Lee et al., 2003; Simmons et al., 2001), persistent production of type I and II interferons driven by activation of toll-like receptors with HIV-1 RNA (Sereti and Altfeld, 2016), fibrosis of lymphoid structure linked to CD4⁺ T cells depletion (Beignon et al., 2005) and mucosal barriers damages associated with microbial translocation (Klatt and Brenchley, 2010; Gordon et al., 2010). Although inflammation can contribute to the early control of HIV infection, immune dysfunction and chronic inflammation can in turn promote viral spread and the replenishment of the reservoir by stimulating the migration of CD4⁺ T cells to sites of viral replication (Klatt and Silvestri, 2012; Hunt et al., 2011), generating new activated target CD4⁺ T cells (Biancotto et al., 2008). This further leads to a reduced ability of the immune system to kill infected cells, and an increased cell-surface expression of immune checkpoints which contributes to the survival of infected cells (Hatano et al., 2013; Chomont et al., 2009). Furthermore, its stimulates latently infected cells to proliferate but also to produce the virus (Chun et al., 1998; Cameron et al., 2010). Altogether, inflammation, poor antiviral immune response, and HIV-1 persistence create a pathogenic cycle. Interfering with mechanisms linking inflammation and HIV-1 persistence could contribute to an HIV cure and the management of inflammation-linked comorbidities. Micci et al., 2015 established the proof of concept that targeting chronic inflammation with IL-21, a potent immunomodulatory cytokine, in ART-treated SIV-infected non-human primates could improve disease prognosis by reducing immune activation and the viral reservoir. To achieve HIV cure, as the viral reservoir is set within few days after infection (Lewis, 2016), early initiation of novel treatment is required to block reservoirs establishment and may protect the immune system (Rutstein et al., 2017).

Inflammasomes plays important roles in innate immunity and inflammation as multiprotein signalling platforms sensing pathogens, danger signals, and regulating cellular functions (Broz and Dixit, 2016). Upon recognition of the microorganism or danger signals, certain Pattern Recognition Receptors (PRRs) recognize the presence of unique microbial components, so-called pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), and recruit a multiprotein signalling platform containing the adapter protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) which induces the proteolytic activation of the procaspase-1 (Broz and Dixit, 2016). Subsequently, active caspase-1 initiates the release of pro-inflammatory cytokines such as IL-1β and IL-18 and the induction of pyroptosis, an highly inflammatory form of cell death. HIV-1 was described to induce the formation and action of an inflammasome in several cell types and models (Doitsh et al., 2014; Biancotto et al., 2007; Ahmad et al., 2002; Song et al., 2006; Wiercinska-Drapalo et al., 2004; Granowitz et al., 1995; Pugliese et al., 2002; Ahmad et al., 2018; Guo et al., 2014; Chattergoon et al., 2014; Pontillo et al., 2012; Pontillo et al., 2013; Walsh et al., 2014; Jakobsen et al., 2013). Levels of IL-1β and IL-18 were found to be elevated in HIV-1 infected individuals (Biancotto et al., 2007; Ahmad et al., 2002; Song et al., 2006) and to induce HIV-1 production (Wiercinska-Drapalo et al., 2004; Granowitz et al., 1995; Pugliese et al., 2002). Ahmad et al. found that a higher proportion of blood monocytes from HIV-1 infected patients were ASC specks positive, a hallmark of inflammasome activation (Ahmad et al., 2018).

In innate immune cells, such as monocytes/macrophages (Guo et al., 2014; Chattergoon et al., 2014), dendritic cells (Pontillo et al., 2012; Pontillo et al., 2013) and microglial cells (Walsh et al., 2014), HIV-1 infection results in the activation of an inflammasome involving the PRR nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR)-containing proteins (NLR) family member 3 (NLRP3). Upon activation of NLRP3 inflammasome, pro-IL-1β is processed by activated caspase 1 to form active IL-1β. NLRP3 inflammasome activation requires a priming signal and an activation signal ultimately leading to pro-inflammatory cytokine release and pyroptosis. More specifically, in human monocytes/macrophages, HIV-1 triggers the NLRP3 inflammasome through the detection of HIV-1 RNA by TLRs and the activation of the NF-κB pathway (Jakobsen et al., 2013). Interferon (IFN)-inducible protein 16 (IFI16) and AIM2 are DNA sensor of the AIM2-like receptors (ALRs) family and can form an inflammasome with ASC upon detection of nuclear DNA. IFI16 is also able to sense HIV-1 in macrophages, but it is unknown whether it can activate an inflammasome in such cell type (Jakobsen et al., 2013). Using lymphoid aggregate culture (HLAC) ex vivo systems from fresh human tonsil or spleen tissues, Doitsh et al. revealed how most of CD4⁺ T cells dies during HIV-1 infection (Doitsh et al., 2010). Over 95% of CD4 T cells are non-permissive to HIV-1 infection and accumulates incomplete DNA transcripts in their cytosol. These transcripts are detected by IFI16, a sensor which activates caspase-1, triggers pyroptosis and the release of intracellular content and pro-inflammatory cytokines including IL-1β (Monroe et al., 2014). The role of the inflammasome in early SIV pathogenesis has recently been shown in the impairment of the innate and adaptive immune response (Barouch et al., 2016) thereby facilitating viral replication (Lu et al., 2016).

Altogether, these results strongly suggest that inflammasome activation occurs early during HIV-1 infection, and could be a central mechanism in the early viral escape to the immune system and furthermore, may facilitate the establishment of the pathogenic cycle of HIV-1 reservoirs persistence and inflammation. VX-765, a selective caspase 1 inhibitor, reduces activation and activity of caspase 1 and antagonizes NLRP3 inflammasome assembly and activation in the context of several inflammatory disorders and diseases such as atherosclerosis, sepsis, or alzheimer disease (Jin et al., 2022; Wu et al., 2021; Flores et al., 2020). Therefore, the aim of our study was to investigate the effects of the Caspase-1 inhibitor VX-765 in an humanized mouse model of HIV-1 infection as a therapeutic strategy to reduce HIV-1-induced inflammation and reservoirs establishment.

We showed in the current work, that targeting inflammasome activation early after HIV-1 infection using the caspase-1 inhibitor VX-765 might represent a potential therapeutic strategy to improve CD4⁺ T cell homeostasis, and to reduce viral load and immune activation. Many studies have outlined the implications of inflammasome activation during the acute and chronic phase of HIV-1 infection as recently reviewed by Leal et al., 2020. The activation of inflammasomes in monocytes, macrophages and dendritic cells leads to the release of cytokines and plays an important part in the first line of host defense against HIV-1 (Leal et al., 2020). However, it can also promote viral spread by the recruitment and activation of CD4⁺ T cells (Klatt and Silvestri, 2012; Hunt et al., 2011). Furthermore, the chronic activation of inflammasomes might be a driver of viral reservoir persistence (Biancotto et al., 2008), which in turn can trigger inflammation, creating a pathogenic cycle. Breaking out of this pathogenic cycle could represent an important building block to reduce non-AIDS-related morbidities caused by persisting inflammation during cART (Deeks et al., 2012). To date, despite increased therapeutic interests (Jin et al., 2022; Wu et al., 2021; Flores et al., 2020), no compound targeting the inflammasome has been marketed (Dutartre, 2016). Nevertheless, the caspase 1 inhibitor VX-765 was approved by the Food and Drug Administration for human clinical trials, showed a good safety profile, and represents currently a promising drug to prevent the onset of inflammation in several diseases (Flores et al., 2020).

In this regard, we investigated in a first step whether the humanized mouse model might be a suitable pre-clinical model to evaluate anti-inflammasone therapy through the detailed kinetics of inflammasome activation in tissues from humanized mice. During the early host response to HIV-1 infection in humanized NSG mice, we described a clear bi-phasic activation profile of inflammasome-related genes in bone marrow, lungs, lymph node, and spleen. The rapid inflammasome-related transcriptomic changes indicate an early sensing of HIV-1 infection, before HIV-1 RNA was quantifiable in the tissues and may reflect the host response to the virus challenge. These findings are in agreement with previous studies from HIV-1 infection in humans (Walsh et al., 2014) and SIV infection in rhesus monkeys (Barouch et al., 2016). Interestingly, in the first phase (up to day 3 post-infection), the expression of NLRP3, IL-1β, and IL-18 was significantly upregulated in several organs before viremia was detectable in blood. Of note, in lymph nodes and bone marrow, transcriptomic changes occurred even before detectability of the virus in the respective tissues and despite variable level of human cells engraftment in tissues of humanized mice. The genes induced in the bone marrow included NLRP3, able to respond to a diverse set of stimuli including HIV ssRNA and proteins, the adaptor ASC, bridging NLRs such as NLRP3 to the inflammatory Caspase-1(CASP-1) and the downstream pro-inflammatory cytokines IL-1β and IL-18 indicating the up-regulation of the entire NLRP3 pathway. In the lymp nodes, the induction of an early inflammasome from day 1 and day 3 p.i. on, was evident from the upregulation of the NOD-like receptors NLRP1, NLRP3, AIM2, CASP-1, and IL-1β, indicating an early host response to HIV-1 infection in humanized mice, in agreement with results obtained from the SIV model (Barouch et al., 2016; Lu et al., 2016). After the initial burst we observed a second phase of transcriptomic changes dominated by IFI16 and AIM2 in bone marrow, lymph nodes, and spleen. In response to DNA, IFI16 is able to mediate the induction of IFN-β through the STING pathway (Unterholzner et al., 2010) but it can also form an inflammasome with ASC upon detection of nuclear DNA (Kerur et al., 2011). AIM2 is a cytoplasmic double stranded DNA sensor that can initiate the assembly of the inflammasome (Hornung et al., 2009; Bürckstümmer et al., 2009; Fernandes-Alnemri et al., 2009). Previous reports indicated that IFI16 restricts HIV-1 infection trough STING in macrophages (Jakobsen et al., 2013) but promotes HIV-1 induced CD4⁺ T cells death by pyroptosis and thus may drive HIV-1 pathogenesis (Doitsh et al., 2010; Monroe et al., 2014). Here we show several direct correlations between IFI16 but also AIM2 expression with viral load and the inverse correlations with the percentage of CD4⁺ T cells (Figure 2). These results are reinforced by several correlations obtained between ASC and CASP-1 expression and the percentage of CD4⁺ T cells in the bone marrow, lymph nodes or in the spleen or with viral load for ASC in the bone marrow 24 days post-infection (Figure 2—figure supplement 1). Taken together these data emphasize the dual role of inflammasome at the onset of HIV-1 infection that fights first the virus and then fuels disease progression.

Inflammasome activation by NLRP3 is a tightly regulated process requiring signals in two main steps. First a priming signal which leads to transcription of inflammasome-related genes and secondly, a signal initiating the assembly of the multiprotein complex. As a result, caspase-1 activity promotes the proteolytic cleavage of the pro-inflammatory cytokines IL-1β and IL-18. We report here significant increases of circulating protein levels of IL-18 at day 10 and 24 post-HIV-1 infection (Figure 3) indicating a full blown inflammasome activity in humanized mice. We also found a high systemic burst of several plasma cytokines (IFN-γ, TNF-α, IP-10, and IL-18) in agreement with the charateristic cytokine storm of acute HIV-1 infection in humans. For IL-1β, we did not find a detectable and significant increase of the cytokine in the plasma of HIV-1 infected mice as compared to non-infected mice, in contrast to mRNA expression in all tissues. This discrepancy might be due to the short half-life of the cytokine (Stacey et al., 2009) and the low basal secretion of protein expression in plasma of the mice. In our humanized mice model, we used an improved engraftment protocol with busulfan myeloablation to reach T cell counts sufficient to sustain long-term HIV replication. Increased CD3⁺ T cell engraftment levels were obtained, constituted of around 61% of human CD3⁺ thymocytes, 8% of human monocytes in the bone marrow, and 3% of human myeloid DCs in the spleen, 22 weeks after engraftment of mice (Singh et al., 2012). Since IL-1β is mainly produced by macrophages and macrophage-like cells, it is tempting to speculate that less protein is secreted after HIV infection in this model as compared to other animal models. In addition, the cytokine is active at the low ng/mL range or even the pg/mL range in humans or in mice. This low level of secretion could explain its fast consumption or fast degradation in plasma of the humanized mice. In contrast, a clear accumulation of IL-18 was shown, and a clear correlation was however achieved between the level of circulating IL-18 and HIV-1 pathogenesis (viral load and loss of CD4⁺ T cells, Figure 3). In summary, all these data demonstrate that the humanized mouse model of HIV infection recapitulates the mean features of inflammasome activation and is well adapted to assess anti-inflammasome therapies against HIV-1-induced immune activation.

To explore the potential effects of direct inflammasome inhibition in vivo, we next evaluated the caspase-1 inhibitor VX-765 administrated as early as day 2 post-infection and for 21 consecutive days. Daily administration of VX-765 significantly reduced circulating levels of IL-18 and levels of circulating pro-inflammatory cytokines, preserved slightly CD4 +T cell homeostasis, and lowered plasma viral load and HIV-1 reservoirs in human CD45⁺ immune cells from the spleen. Interestingly, since a low CD4/CD8 T cell ratio is associated with disease progression to AIDS, our results suggest that VX-765 treatment may delay disease progression and preserve CD4⁺ T cell homeostasis. Pyroptotic CD4⁺ T cells in lymphoid organs was shown through in vitro models to release pro-inflammatory cytokines, inducing a local inflammation that further recruits new CD4⁺ T cells to the site of infection. In addition, innate immune cells recruitment and the activation of an NLRP3 inflammasome might enhance the release of pro-inflammatory mediators. This globally enhances HIV-1 replication, thus stimulating the replenishment of the reservoir. It is worthy to note that treatment with VX-765 reduced significantly plasma viral load and the formation of the HIV-1 reservoir in the spleen of humanized mice. This result needs to be further confirmed in central memory and effector memory T cells in a model of HIV-1 latency in humanized mice in combination with cART treatment to explore this potential.

We show here that inflammasome activation in vivo rather takes places in splenic innate immune CD11c⁺ and CD14⁺ cells than in the splenic adaptive CD4⁺ or CD8⁺ T cells or in the bone marrow immune cells indicating that HIV-1 induced inflammasome activation in splenic CD11c⁺ cells may be the principal source of circulating IL-18. When investigating pyroptosis in subsets of splenic caspase-1 activated cells, we did not find any significant increase in cell death in CD4⁺ and CD8⁺ T cells. In addition, we did not detect any effect of VX-765 in caspase-1 activation, suggesting that the small caspase-1 activation observed in these subsets might reflect a background activity and could probably be induced earlier by HIV-1 infection, as proposed by Terahara K et al. who showed interestingly that live caspase 1 cells might rapidly die in humanized mice (Terahara et al., 2021). Our in vivo data did not confirm previous results obtained in vitro only by the group of Doitsh et al., 2010 who proposed pyroptosis as the main mechanism leading to CD4⁺ T cell depletion after HIV-1 infection. We can not exclude that the assay is not sensitive enough in primary human T cells from humanized mice to show pyroptosis or that we did not catch an earlier best time point. In addition, there is no evidence yet that such microenvironment of abortive infected and bystander CD4⁺ T cells can be recapitulated in lymphoid organs of humanized mice where fewer human CD4⁺ T cells remains as compared to human lymphoid organs. This could also explain the minimal effect of VX-765 on CD4 ⁺T cell depletion as compared to previous results obtained in vitro by Doitsh et al., 2014 HIV-1 infection is associated with programmed cell death, and NLRP3 inflammasome-mediated immune T cell depletion was nevertheless shown here by different correlations with the dowstream pathways of NLRP3. Through serial necropsies, Barouch et al. demonstrated that, following SIV infection, early host response at the mucosal portal of entry and early sites of distal virus spread involved a robust expression of components of the inflammasome and TGF-β pathways which impairs both innate and adaptative immunities (Barouch et al., 2016). Other anti-inflammatory strategies aiming at breaking this vicious circle and reducing HIV persistence are currently investigated in several clinical trials, including the anti-inflammatory, anti-fibrotic angiotensin II blocker losartan (phase 2, NCT01852942) and angiotensin II receptor antagonist telmisartan (phase 1, NCT02170246), immunosuppressors with mTOR inhibitory activities such as sirolimus (phase 2, NCT02440789) and everolimus (phase 4, NCT02429869) or the anti-inflammatory janus kinase inhibitor ruxolitinib (phase 2, NCT02475655). Most of these strategies target inflammation during chronic HIV infection. HIV-1-induced massive systemic immune activation was repeatedly linked to poor outcomes during chronic infection such as the increased risk of cardiovascular complications (Ross et al., 2009; Graham et al., 2013). Furthermore, levels of cytokines IP-10 and IL-18 remain elevated in cART patients compared to healthy donors and elite controllers (Côrtes et al., 2018). Therefore, complementary approaches are needed to reduce persisting immune activation under cART; inhibiting early inflamasomme activation might be a very promising approach that could be combined to cART for treating acute HIV infection. During chronic HIV-1 infection under cART therapy, residual viral replication and viral protein expression from defective provirusses is a continuous driver of inflammation and might also trigger inflammasome activation (Klatt and Silvestri, 2012; Hunt et al., 2011; Biancotto et al., 2008). Furthermore, the incapacity of cART to completely restore immune functions and control chronic inflammation promotes immune aging and the emergence of non-AIDS-related diseases such as cancer, cardiovascular diseases, viral infections, and cognitive decline (Deeks et al., 2012). Although cART reduce the aberrant cytokine production of monocytes, some inflammatory monocytes subsist while phagocytic monocytes decrease, resulting in a lower antigen presentation to CD8⁺ T cells (Novelli et al., 2020). Taken together, all these data suggest that inflamasomme inhibition, by offering simultaneous reduction of inflammation and affecting HIV-1 replication, might be further evaluated to support cure research.

The data have been uploaded in the Zenodo repository.

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Decision letter after peer review:

Thank you for submitting your article "The anti-caspase 1 inhibitor VX-765 reduces immune activation, CD4 + T cell depletion, viral load and total HIV-1 DNA in HIV-1 infected humanized mice" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Satyajit Rath as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The study examined the induction of inflammasome activation by HIV infection in a humanized NSG mouse model. The results suggest that inflammasome activation plays a key role in CD4 T cell depletion and can be inhibited by the anti-caspase 1 inhibitor VX-765. The results are suggestive but not all findings seem entirely consistent and some issues regarding the correlation analyses, significance of some cytokine measurements, and inability to detect caspase-1 activity in CD4 and CD8 T cells need to be addressed.

Essential revisions:

1. How do the authors explain lack of plasma viremia despite high levels of HIV RNA in all tissues and blood at day 10 in two of the animals. Please also address the inconsistencies in inflammasome gene and IL-18 upregulation across tissues and timepoints noted by reviewer 1.

2. Are the correlations shown in Figure 2 (and figure S4) still significant if only infected animals are included?

3. The levels of some cytokines shown in figure 3 and figure 4 seem to be below the usual limit of detection for the assay used. This should be clarified and only reliable measurements included in correlation analyses.

4. It is surprising that no caspase-1 activity could be detected in CD4 and CD8 T cells. The authors should try to use a more sensitive assay, if available. If caspase-1 activity is not increased in CD4 T cells after HIV infection in this mouse model differences to previous data using human tissues and the overall hypothesis that inflammasome activity contributes to viral persistence in CD4 T cells need more discussion.

5. The protective effect on CD4 T cells is modest and should be presented with caution. In addition, the discussion about HIV reservoirs should be shortened or (better) the effect of VX-765 on the HIV reservoirs e.g. in central and effector memory CD4 T cells should be determined to strengthen this part.

6. It should be examined whether administration of the anti-caspase 1 inhibitor VX-765 suppresses the induction of inflammasome-associated genes upon HIV infection in this mouse model.

Reviewer #1 (Recommendations for the authors):

Specific comments:

1. The manuscript contains numerous typos and grammatical errors and should be extensively spellchecked before resubmission.

2. It is surprising to see high levels of HIV RNA in all tissues and blood at day 10 but no detectable plasma viremia in 40% (2 out of 5) of the animals. The authors should comment on this discrepancy.

3. There appear to be inconsistencies in inflammasome gene upregulation across tissues and timepoints. Is there a biological explanation for this or is it due to inconsistencies in the qPCR assay? Furthermore, IL-18 gene transcription only appears to be upregulated in the BM at D3 post infection and in none of the other tissues, particularly at D24 post-infection. This seems quite different from the cytokine levels measured in Figure 3, where IL-18 is 10-fold higher at D24 than at pre-infection. What would be the reason for this discrepancy?

4. It is shown clearly that IFI16 and AIM2 expression are increased after infection, and this fits with the hypothesis and previous literature showing inflammasome activation after HIV infection. However, the correlations in Figure 2 (and figure S4) appear quite misleading, as they seem to be solely driven by the inclusion of data from uninfected animals. These correlations need to be shown only including infected animals, as there is a large enough heterogeneity in the viral loads and CD4 counts of these animals to appreciate any true associations.

5. There appear to be issues with the measurement of a number of the cytokines shown in figure 3 and figure 4. While the authors do not describe the limit of detection for the assay used, they indicated they are using Mesoscale U-PLEX biomarker assay kit. The listed LOD for this kit includes IL-1B at 0.15 pg/mL, IL-6 at 0.33 pg/mL and TNF-a at 0.51 pg/mL. It appears that the large majority, if not all, of the measurements of these 3 cytokines are below the limit of detection. To display these data points as accurate values without any indication of this is incorrect. If this is the true LOD, all data points below that limit should be clearly indicated and should not be used in correlation analyses without imputed values. If the authors have an alternative LOD than that listed for the commercially available kit, it needs to be made clear what that limit is and how it was calculated. It is concerning that the primary cytokine involved in caspase-1 activation of the inflammasome (as described in line 103) is unable to be measured in this manuscript.

6. Figure 5 could be improved by clearly indicating the day post-infection the data for panels B, C, E and F represent. Furthermore, panel E in figure 5 is indicated to show Blood in the y axis label, but BM in the figure legend.

7. The authors should clarify the issue with the lack of caspase-1 activity in CD4 and CD8 T cells detected. If this is a problem with the assay, a different approach needs to be used. If caspase-1 activity is not increased in CD4 T cells after infection in their model, this data impacts the overall hypothesis that inflammasome activity contributes to viral persistence in CD4 T cells and requires a larger discussion.

8. The impact on CD4 T cell preservation seems minimal. While there is a statistically significant difference in the blood, it appears to be very slight. Furthermore, this difference does not extend to the spleen. This should be acknowledged.

9. The Y axes in figure 6 panels A, B, and C should all be shown in log scale, with no split axes and should all be equivalent to each other as is standard for displaying plasma viral loads in HIV studies.

10. There is much discussion of the importance of limiting the HIV reservoir and doing so in combination with early-initiated antiretroviral therapy. This discussion should be reduced, as this manuscript does not follow animals beyond day 22 post-infection and does not include animals receiving ART.

Reviewer #2 (Recommendations for the authors):

1. The Authors explained well in the introduction the elimination of HIV reservoirs as a key factor for HIV cure. Also, the Authors reported anti-caspase 1 inhibitor VX-765 reduces HIV reservoirs. However, in the study the Authors did not quantify the different HIV reservoirs (For example Central memory and effector memory CD4 T cells) and the effect of VX-765 on the population of these HIV reservoirs. It will be helpful if the authors could illustrate if VX-765 reduces the population of HIV reservoirs

2. The authors nicely used a negative control for the anti-caspase 1 inhibitor VX-765 during the study. However, there was no positive control. The design of the experiment can be improved by including a positive control like anti-inflammatory drug.

3. The authors nicely illustrated the expression of genes associated with inflammasome activation in HIV infection. However, this was not done after administration of the anti-caspase 1 inhibitor VX-765. It will make more sense to also illustrate the changes in the transcriptomic profile of markers of inflammasome activation after treatment with VX-765.

4. Studies have revealed that bystander CD4 T pyroptosis account for about 95% depletion of CD4 T cells in HIV infection https://doi.org/10.1016/j.cell.2010.11.001. However, in this study it was shown that there was more inflammasome activation in infected than uninfected CD4 T cells (Figure 2). Could the Authors expand more of this?

Reviewer #1 (Recommendations for the authors):

Specific comments:

1. The manuscript contains numerous typos and grammatical errors and should be extensively spellchecked before resubmission.

The manuscript has been reviewed and typos and grammatical errors have been corrected.

2. It is surprising to see high levels of HIV RNA in all tissues and blood at day 10 but no detectable plasma viremia in 40% (2 out of 5) of the animals. The authors should comment on this discrepancy.

As explained in the section Material and Methods, HIV-1 mRNA was measured in mice using plasma (Figure 1—figure supplement 1) and using RNA extracted from CD45 + cells isolated from tissues or blood (Figure 1—figure supplement 2). For plasma viral load, a maximum of 30 µl whole blood was collected from the temporal vein for longitudinal sampling. 30 µl of mice plasma were diluted in 70 µl of PBS for RNA extraction and RNA extracted as described in P Adams et al. (iScience Volume 24, Issue 1, 22 January 2021). Human CD45 + cells were isolated from peripheral blood (collected by cardiac puncture at necropsy) or spleen, bone marrow and lymph nodes single cell suspensions using CD45 MicroBeads, and total RNA was extracted using an input of 100 µl of cell lysates after purification. Therefore, the input for RNA extration is much lower in plasma than in cell lysates or in blood and this explain the discrepancy between the two measurements. This is now detailed in the revised material and methods. In addition, we used a protocol of humanisation with busulfan myeloablation regimen and transplantation of CD34 + cell isolated from cord blood in 3–4 week old NSG mice. As shown in Author response image 1, the level of engraftment was more important in tissues than in peripheral blood (human CD45 ⁺ cells measured by flow cytometry when organs were collected). This explain why HIV-mRNA was detected earlier in human CD45 + isolated from tissues than from blood as soon as day 3. The 2 animals that displayed no detectable viremia at day 10 showed also less HIV-1 mRNA in human CD45 ⁺ cells isolated from blood and tissues (NS127 and NS136). Our results are in agreement with Barouch et al. (Cell 2016) which reported a similar earlier HIV-1 mRNA detectability in tissues as compared to blood in a SIV infection model using Rhesus monkeys.

Author response image 1

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3. There appear to be inconsistencies in inflammasome gene upregulation across tissues and timepoints. Is there a biological explanation for this or is it due to inconsistencies in the qPCR assay?

Inflammasome upregulation was measured at day 1, 3, 10 and 24 after HIV infection from human CD45⁺ cells isolated from the different tissues coming from different mice pooled for each day of measure (D0, D1, D3, D10, D20) and sacrificed. Therefore, the results obtained at day 1, 3 , 10 or 24 are not longitudinal samples coming from the same mice but from different mice and could explain the variabiliy and inconsistencies in inflammasome gene upregulation. Indeed, HIV-1 replication is quite variable among the mice and induced a loss of CD4⁺ T cells that is also variable among the tissues and mice in time (as shown in Author response image 2). This is also the case for the number of CD8⁺ T cells in tissues from the different mice (Author response image 3). This number is variable and based on the level of engraftment of each mice in each tissue, similarly to the other human immune cells producing the different inflamasomme genes or cytokines. All these reasons can explain the inconsistencies observed in Figure 1—figure supplement 3 of the manuscript that are not due in the qPCR assay.

Author response image 2

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Author response image 3

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Furthermore, IL-18 gene transcription only appears to be upregulated in the BM at D3 post infection and in none of the other tissues, particularly at D24 post-infection. This seems quite different from the cytokine levels measured in Figure 3, where IL-18 is 10-fold higher at D24 than at pre-infection. What would be the reason for this discrepancy?

Il-18 has unique characteristics that could explained why we have observed only an early upregulation of its mRNA at day 3 in the bone marrow and then a net accumulation in plasma untill day 24. The cytokine is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine. Importantly, in contrast to IL-1β, the IL-18 precursor is constitutively present in nearly all cells in healthy humans and animals (Novick D et al., Interleukin-18, more than a Th1 cytokine, Seminars in Immunology, 2013, Volume 25, Issue 6). Many cell types produce IL-18 including hematopoietic cells and circulating monocytes, resident macrophages, and DCs. The IL‐18 precursor is also found to be released by endothelial cells, keratinocytes, osteoblasts, most intestinal epithelial cells throughout the entire gastrointestinal tract, and mesenchymal cells. IL‐18 can also be discharged in its precursor form from dead cells which can be acted upon by neutrophil proteases such as proteinase most 3 into its active form (Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment, Amarachi Ihim S. et al. Front. Immunol., 11 August 2022). Peritoneal macrophages and mouse spleen also contain the IL-18 precursor. In the absence of disease, the IL-18 precursor is constitutively expressed in endothelial cells, keratinocytes and intestinal epithelial cells. Macrophages and dendritic cells are the primary sources for the release of active IL-18, and this could explain why we found only an early mRNA upregulation of Il-18 in the bone marrow coming from hematopoietic cells and then the IL-18 cytokine is released in plasma upon activation by the cells containing the constitutive precursor. Indeed, IL-18 requires 10–20 ng/mL and sometime higher levels to activate target cells as compared to IL-1β that is active on cells in the low ng/mL range and often in the pg/mL range (Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment, Amarachi Ihim S. et al. Front. Immunol., 11 August 2022). Taken together, these data support the strike difference between IL-18 and Il-1β concentrations measured in humanized mice after HIV infection and the fact that Il-18 accumulated in plasma and not at the gene level in the spleen or the lymp nodes. A sentence were added in the results part of the manuscript and in the discussion to explain this.

4. It is shown clearly that IFI16 and AIM2 expression are increased after infection, and this fits with the hypothesis and previous literature showing inflammasome activation after HIV infection. However, the correlations in Figure 2 (and figure S4) appear quite misleading, as they seem to be solely driven by the inclusion of data from uninfected animals. These correlations need to be shown only including infected animals, as there is a large enough heterogeneity in the viral loads and CD4 counts of these animals to appreciate any true associations.

The correlations have been now performed by including the mice showing evidence of infection with detectable HIV-RNA in tissues or in blood, and several correlations remained significantly significant. Figure 2 and Figure 2—figure supplement 1 have been updated accordingly with the new graphs of correlations.

5. There appear to be issues with the measurement of a number of the cytokines shown in figure 3 and figure 4. While the authors do not describe the limit of detection for the assay used, they indicated they are using Mesoscale U-PLEX biomarker assay kit. The listed LOD for this kit includes IL-1B at 0.15 pg/mL, IL-6 at 0.33 pg/mL and TNF-a at 0.51 pg/mL. It appears that the large majority, if not all, of the measurements of these 3 cytokines are below the limit of detection. To display these data points as accurate values without any indication of this is incorrect. If this is the true LOD, all data points below that limit should be clearly indicated and should not be used in correlation analyses without imputed values. If the authors have an alternative LOD than that listed for the commercially available kit, it needs to be made clear what that limit is and how it was calculated.

The LOD of the Mesoscale assay (Author response table 1, LOD of the manufactor) have been now added in the material and methods and all the measures being under the LOD have been excluded in the analyses. Therefore, only the graphs and correlations with a sufficient number of detectable measures for statistical analyses are now shown in Figure 3, Figure 3—figure supplement 1, Figure 4, and Figure 4—figure supplement 1.

It is concerning that the primary cytokine involved in caspase-1 activation of the inflammasome (as described in line 103) is unable to be measured in this manuscript.

As mentioned above, IL-1β activate target cells in the low ng/mL range and often in the pg/mL range (Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment, Amarachi Ihim S. et al. Front. Immunol., 11 August 2022) in human or animals. In addition, in healthy human subjects and also in healthy mice, gene expression for IL-1β in blood mononuclear cells and hematopoietic cells is absent and there is no evidence that the IL-1β precursor is constitutively present in epithelial cells (Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment, Amarachi Ihim S. et al. Front. Immunol., 11 August 2022). To our knowledge, the MSD assay was the most sensitive and quantitative available test (dynamic range between 0.15 and 3.8 pg/ml), but was not sensitive enough to catch a detectable and significant increase of Il-1β in humanized mice infected with HIV. This could be a combined consequence of a very low level of secretion of IL-1β by the engrafted human cells and the short half life of IL-1β in plasma of the mice. The cytokine being active at the low ng/mL range or even the pg/mL range, this could explain its fast consumption or degradation in plasma. We also tried western-blotting for Il-1 β, but we could not retrieve enough of proteins from human CD45+ cells isolated from the tissues of the humanized mice to evaluate its expression.

We added two sentences in the result part and in the discussion part to clarify the absence of IL-1β in plasma of the mice.

6. Figure 5 could be improved by clearly indicating the day post-infection the data for panels B, C, E and F represent. Furthermore, panel E in figure 5 is indicated to show Blood in the y axis label, but BM in the figure legend.

The figure 5 has been changed accordingly and mention now Day 22 post-infection above each graph. Panel E is indeed the panel of bone marrow, the name Blood was an error and was changed to BM in the revised Figure 5.

7. The authors should clarify the issue with the lack of caspase-1 activity in CD4 and CD8 T cells detected. If this is a problem with the assay, a different approach needs to be used. If caspase-1 activity is not increased in CD4 T cells after infection in their model, this data impacts the overall hypothesis that inflammasome activity contributes to viral persistence in CD4 T cells and requires a larger discussion.

To measure the Caspase-1 activity in the bone marrow and lymph nodes of humanized mice, we used the fluorescent labeled inhibitors of caspase-1 (FLICA) technique to stain active caspase -1 with a FAM probe. We used the same probe as in the work of Doitsh G. et al. in 2014 who demonstrated the role of caspase-1 in ex vivo human lymphoid aggregate culture (HLAC) system formed with fresh human tonsil or spleen tissues, to reflect HIV-1 infection by a small number of viral particles without mitogens in a lymphoid microenvironment. Infection of these cultures with HIV-1 produces extensive loss of CD4 T cells, but, as described by the authors, >95% of the dying cells are abortively infected with HIV reflecting their nonpermissive, quiescent state. Flow cytometry is the most sensitive tool that we could use in our humanized model of HIV-1 infection to detect caspase-1 activity. The Caspase-Glo 1 Inflammasome Assay (Promega) was tested but was not sensitive enough to detect a sufficient caspase 1 activity level in human PBMCs isolated from bone marrow or the lymph nodes of the mice. We do not think that the assay is a problem but we rather missed an early time point that would have been the best for T cells since we focused our analysis on the primary end point of viral load, 22 days after HIV infection.

In our experiments, we used lymphocytes isolated from the blood or lymphoid tissues that are probably mostly permissive to HIV infection and give rise to new virions (Cooper et al., 2013; Gougeon et al., 1996). However, in human lymphoid tissues such as tonsil and spleen, HIV enters resting non-permissive cells that represent >95% of the CD4 T cell population according to the group of Doitsh et al. (Doitsh et al., 2010; Eckstein et al., 2001; Moore et al., 2004). As suggested by these authors, these non-permissive cells undergo abortive infection and ultimately die due to an innate immune response launched by the host against cytosolic viral DNA culminating in a caspase 1-dependent pyroptosis (Doitsh et al., 2014; Monroe et al., 2014). Doitsch et al. have also used fresh lymph nodes from subjects infected with R5-tropic HIV, where caspase-1 and IL-1β are nested in the paracortical zone rich in resting CD4 T cells using immunohistochemistry. In our model of humanized mice using busulfan treatment, we observed an improved development of lymphoid structures such as lymph nodes, however, at 23 days after HIV-1 infection, the lymph nodes of mice contain only around 40% of human CD4 + T cells (see Author response image 2). In this lymphoid environment, the Caspase 1 inhibitor should not inhibit productive HIV-1 infection but should block pyroptosis of abortively infected CD4 T cells (Doitsh et al., 2014) mediated by cell-to-cell spread culminating in the pyroptotic death on nonpermissive CD4 T cells. Most of the pathogenic effects of HIV-1 were not attributed to killing of CD4 T cells by circulating free virions but by cell-to-cell contact (Galloway N LK, Cell report 2015). Although several mechanisms of HIV-1 mediated pathogenesis proposed in humans can occur in hematopoietic stem cells-transplanted humanized mice, the immune system poorly supports adaptive immune responses due to the lack of HLA expression in the mouse thymus. At first, there is no evidence yet that such microenvironment with abortive infected CD4 T cells can be reproduced in the lymph nodes or the spleen of humanized mice. However, 22 days after HIV infection, we observed caspase-1 activity only in CD11c⁺ and CD14 ⁺ cells but not in CD4⁺ or CD8 ⁺ T cells. It is important to note, that only the group of Doitsh et al., using a HLAC microenvironment, was able to demonstrate these results. These results were not reproduced by other groups. It is tempting to speculate that, similarly, we were not able to reproduce these results, because we are not using the same type of culture with fresh isolated organs preserving the non permissive and dying CD4 T cells, and therefore we did not catch caspase 1 induced pyroptosis in T cells isolated from the humanized mice.

In addition, a recent study in humanized mice, published in 2021 after the realisation of our experiments suggest that cell death of both naïve and memory CD4 T cells is induced in the spleen at 3 days after the HIV-1 challenge and before the significant loss in peripheral blood (Substantial induction of non-apoptotic CD4 T-cell death during the early phase of HIV-1 infection in a humanized mouse model, Kazutaka Terahara et al., Microbes and infection 2021). The authors used the FLICA technique, and even though dead cells proceeded more degradation of HIV components and direct infection induced cell death, the authors observed that, within live CD4 T cells, Caspase 1 positive cells had more viral reverse-transcripts, suggesting Caspase 1 as an early marker for HIV infection. Furthermore, since the percentages of Caspase 1 positive cells within live CD4 T cells did not significantly increase following HIV challenge (Supplementary Figure 2), the authors suggest that live caspase 1 cells might rapidly die. The authors observed that most dead CD4 T cells were HIV negative in vivo in humanized mice. While these HIV-negative dead cells might have contained cells in which HIV components were degraded, the other cells might have undergone bystander cell death, which is induced when neighboring cells undergo inflammatory cell death, such as pyroptosis and necroptosis, and release proinflammatory mediators that stimulate more cells to die. Therefore, we probably missed the early caspase 1 activity in T cells and just caught the caspase 1 activity in CD11c⁺ and CD14 ⁺ cells that occurred probably later in time in the spleen.

In conclusion, we can assume that caspase 1 positive T cells are dying fast, and that we were not able to detect them, since the number of human T cells in humanized mice is also limited. In our analysis, we used a live dead staining to discriminate between live and dead cells based on membrane integrity. The Live-dead positive cells showed some FLICA staining, as mentionned in Figures 8, 9, and figure 9—figure supplement 1, and VX-765 diminished significantly the number of FLICA positive cells in splenic CD11c⁺ and CD14⁺ cells but not in T cells, where ony residual FLICA staining seems to occur. We analysed the number of FLICA positive cells among the “debris” from the spleen in the flow scatters of the experiment of Figure 8 and Figure 9 for each mouse and identified a level of Caspase 1 activity in the cellular debris of the samples in advanced status of death or necrosis. We observed a mean of 11.6 ±0.8 % ( ± SD) of debris positive for FLICA in Control mice versus 5.1 ± 0.7% (p< 0.05) in VX-765 treated mice, as shown in Author response image 4 for two representative mice. These data indicated that a higher number of Caspase 1 positive cells were found in the cellular debris of HIV-infected huNSG mice as compared to VX-765 treated mice, and could be due to some specific staining to T cells in advanced stage of death. Therefore, we globally assume that we could have missed an early time-point to demonstrate the effects of the caspase-1 inhibitor on dying or pyroptic T cells. However, at 22 days after HIV infection, caspase 1 activity was shown to be mainly decreased by the caspase-1 inhibitor in splenic CD11c⁺ and CD14 ⁺ cells.

Author response image 4

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We added a sentence in the discussion part to clarify that we probably missed pyroptosis of T cells that occurred early in HIV infection as proposed by Kazutaka Terahara et al.

8. The impact on CD4 T cell preservation seems minimal. While there is a statistically significant difference in the blood, it appears to be very slight. Furthermore, this difference does not extend to the spleen. This should be acknowledged.

We agree with the reviewer that the impact of VX-765 is minimal, in contrast to the results of Doitsh et al. This was added in the results and the discussion parts.

It was shown that caspase 1 inhibitor did not inhibit productive HIV-1 infection but block pyroptosis of abortively infected CD4 T cells (Doitsh et al., 2014). In our humanized mice model, although we probably missed the CD4⁺ T cells pyroptosis at earlier time points, the effect of the caspase 1 inhibitor in CD4⁺ T cells depletion was also limited. The fact that the effect of VX-765 is weak together with an early inititiation of pyroptosis that was not sustainable at 22 days after HIV infection, could indicate that the humanized mice model failed to recapitulate a microenvironment leading to T cells pyroptosis. Specifically, cell-to-cell spread of HIV-1 was shown to be required to deplete non-permissive lymphoid CD4 T cells via caspase 1-dependent pyroptosis. This phenomenon can be reduced in an environment where human and murine cells co-exist and only 40% of human T cells are present.

It is important to add again that only one research group (Doitsh et al., 2014) described caspase 1-mediated pyroptosis triggered by abortive viral infection in lymphoid tissues following HIV infection using HLACs. They also demonstrate that the mode of HIV-1 spread determines the outcome form of cell death. The productively infected cell ultimately dies by apoptosis, while the bystander resting cell dies by pyroptosis. Since the killing of CD4⁺ T cells is not attributed to circulating free virions but to infected cells residing in lymphoid tissues that mediate cell-to-cell spread of the virus linked in a single pathogenic cascade, we can assume that the balance between productive and abortive infections could be disturbed in our humanized mice model and did not ensure a rapid depletion of CD4⁺ T cells through pyroptosis. Productively infected cells are obligatorily required to transmit the virus across the virological synapse formed with resting CD4 T cells, and they are decreasing with time in our model, probably faster than in human.

Taken together, all these arguments can justify that fewer bystander resting cells are present in the cells isolated from our mice (as compared to the HDAC model of Doitsh et al) and explain a lower effect of VX-765 CD4⁺ T cell depletion. This was added in the discussion part.

9. The Y axes in figure 6 panels A, B, and C should all be shown in log scale, with no split axes and should all be equivalent to each other as is standard for displaying plasma viral loads in HIV studies.

The changes of the Y axes in log scale, with no split, have been done for Figure 6 panels A, B and C.

10. There is much discussion of the importance of limiting the HIV reservoir and doing so in combination with early-initiated antiretroviral therapy. This discussion should be reduced, as this manuscript does not follow animals beyond day 22 post-infection and does not include animals receiving ART.

We agree with the reviewer and we removed the discussion on HIV reservoirs.

Reviewer #2 (Recommendations for the authors):

1. The Authors explained well in the introduction the elimination of HIV reservoirs as a key factor for HIV cure. Also, the Authors reported anti-caspase 1 inhibitor VX-765 reduces HIV reservoirs. However, in the study the Authors did not quantify the different HIV reservoirs (For example Central memory and effector memory CD4 T cells) and the effect of VX-765 on the population of these HIV reservoirs. It will be helpful if the authors could illustrate if VX-765 reduces the population of HIV reservoirs

In this work, we wanted to primarly assess the different effects of caspase-1 inhibition on HIV-1 infection. Our model is a model of HIV-1 infection and not a model of HIV-1 latency by treating animals with cART thereby preserving CD4⁺ T cell depletion. Since CD4⁺ T cells are depleted overtime by HIV-1 infection in the current mice model, we measured the effet of VX-765 only in total HIV-1 DNA isolated from total human CD45+ cells to have enough DNA for quantification. We have previously developed an HIV-1 latency model in humanized NSG mice (P Adams et al., iScience 2021) to investigate the early establishment of the viral reservoir with cART. This model is more appropriate to evaluate the effects of caspase inhibition associated with early cART on viral reservoirs and could better decipher the effects of caspase inhibition in the different memory subsets of T cells. As suggested by reviewer 1, we have decreased the size of the discussion on viral reservoirs, and would suggest to evaluate the effects of inflammasomme inhibitors in central memory or effector memory CD4⁺ T cells in a future work with the latency model of HIV-1 infection.

2. The authors nicely used a negative control for the anti-caspase 1 inhibitor VX-765 during the study. However, there was no positive control. The design of the experiment can be improved by including a positive control like anti-inflammatory drug.

In a previous work, we have published the effect of minocycline, an immunomodulator, in our humnaized mice model, that attenuates HIV-infection and chronic immune activation (M Singh et al., Immunology. 2014 Aug; 142(4): 562–572. Minocycline attenuates HIV-1 infection and suppresses chronic immune activation in humanized NOD/LtsZ-scidIL-2Rγnull mice). We showed that treatment of minocycline at a dose of 100 mg/kg/day for 60 days led to significantly lower viral load and improved T-cell count. Expression of cellular activation (CD38, HLA-DR, CD69 and CCR5) and exhaustion markers (PD-1 and CTLA-4) was significantly lower in the treated group of mice. We knew that the humanized NSG mouse model was able to recapitulate the HIV-induced chronic immune activation observed in infected humans. Since we observed at first a nice up-regulation of the genes involved in inflammasome activation, we did not choose therefore to use a concommittent positive control. Instead, we measured the expression of certain activation markers in CD4⁺ and CD8 + T cells (CD38, CD69, PD-1, HLA-DR) in control and VX-765 treated groups but we did not find any significant differences between the two groups, only a trend displayed in Author response image 5 when the expression of the tree markers was combined.

Author response image 5

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3. The authors nicely illustrated the expression of genes associated with inflammasome activation in HIV infection. However, this was not done after administration of the anti-caspase 1 inhibitor VX-765. It will make more sense to also illustrate the changes in the transcriptomic profile of markers of inflammasome activation after treatment with VX-765.

Using left-over RNA extracted from human CD45+ cells isolated from the spleen of 5 mice in the Vehicle group and 5 mice in the VX-765 group, at 11 and 22 days after HIV-infection, we measured and normalized the expression of AIM2, IFI16, ASC, and CASP-1mRNA. Treatment with VX-765 reduced significantly their expression at both time points. The results are now added in the manuscript in the Figure 4—figure supplement 1E, F, G, H.

4. Studies have revealed that bystander CD4 T pyroptosis account for about 95% depletion of CD4 T cells in HIV infection https://doi.org/10.1016/j.cell.2010.11.001. However, in this study it was shown that there was more inflammasome activation in infected than uninfected CD4 T cells (Figure 2). Could the Authors expand more of this?

We agree with the reviewer and we think that this remark is very important.

As explained for Reviewer n°1, a recent study in humanized mice, published in 2021 after the realisation of our experiments suggest that cell death of both naïve and memory CD4 T cells is induced in the spleen at 3 days after the HIV-1 challenge and before the significant loss in peripheral blood (Substantial induction of non-apoptotic CD4 T-cell death during the early phase of HIV-1 infection in a humanized mouse model, Kazutaka Terahara et al., Microbes and infection 2021). The authors used the FLICA technique, and even though dead cells proceeded more degradation of HIV components and direct infection induced cell death, the authors observed that, within live CD4 T cells, Caspase 1 positive cells had more viral reverse-transcripts, confirming our data and suggesting that bystander CD4 T pyroptois might happen early during HIV infection. Furthermore, since the percentages of Caspase 1 positive cells within live CD4 T cells did not significantly increase following HIV challenge in this work, the authors suggested that live caspase 1 cells might rapidly die. The authors observed that most dead CD4 T cells were HIV negative in vivo in humanized mice. While these HIV-negative dead cells might have contained cells in which HIV components were degraded, the other cells might have undergone bystander cell death, which is induced when neighboring cells undergo inflammatory cell death, such as pyroptosis and necroptosis, and release proinflammatory mediators that stimulate more cells to die. Therefore, these resuts confirmed that inflamasomme activation occured early in live infected CD4⁺ T cells in our humanized mice model and is then pursued in infected CD11c⁺ and CD14⁺ cells up to 24 days after HIV infection in infected animals. The low caspase 1 activity would indicate that we were not able to recapitulate the bystander effect in humanized mice. We would like also emphasize that the bystander hypothesis was never reproduced so far by other groups of research, other than the group of Doitsh et al. This is now clearly indicated in the discussion.

Author details

1. Mathieu Amand

   Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing – original draft, Project administration

   Competing interests

   No competing interests declared

2. Philipp Adams

   Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg

   Contribution

   Conceptualization, Resources, Formal analysis, Investigation, Writing – original draft

   Competing interests

   No competing interests declared

3. Rafaela Schober

   Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg

   Contribution

   Resources, Data curation, Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Gilles Iserentant

   Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg

   Contribution

   Resources, Formal analysis, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Jean-Yves Servais

   Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg

   Contribution

   Resources, Formal analysis, Writing - review and editing

   Competing interests

   No competing interests declared

6. Michel Moutschen

   Department of Infectious Diseases, University of Liège, CHU de Liège, Liège, Belgium

   Contribution

   Conceptualization, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

7. Carole Seguin-Devaux

   Department of Infection and Immunity, Luxembourg Institute of Health, Esch sur Alzette, Luxembourg

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration

   For correspondence

   carole.devaux@lih.lu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0636-5222

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