key: cord-0025241-04wozetl authors: Daskou, Maria; Mu, William; Sharma, Madhav; Vasilopoulos, Hariclea; Heymans, Rachel; Ritou, Eleni; Rezek, Valerie; Hamid, Philip; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Grijalva, Victor; Chattopadhyay, Arnab; Kitchen, Scott G.; Fogelman, Alan M.; Reddy, Srinivasa T.; Kelesidis, Theodoros title: ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV date: 2022-01-07 journal: PLoS Pathog DOI: 10.1371/journal.ppat.1010160 sha: f65aac4cd80d86580ed298913f3012225abca4a2 doc_id: 25241 cord_uid: 04wozetl Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Despite antiretroviral therapy (ART), chronic treated HIV is a state of systemic inflammation and immune activation [1] . Innate immunity biomarkers of inflammation including cytokines like interleukin-(IL)-1β [2] , IL-6 [3, 4] , tumor necrosis factor-α (TNF-α) [3, 5] , chemokines like C-X3-C Motif Chemokine Ligand 1 (CX3CL1)/Fractalkine [6, 7] and biomarkers of monocyte/ macrophage (M/M) activation such as soluble CD14 (sCD14) and sCD163 [1, 8, 9] are predictors of morbidity in people with chronic treated HIV (PWH) [1] [2] [3] [4] [5] [6] 8, 9] . M/M rather than T cell activation is considered a more clinically relevant predictor of morbidity in chronic treated HIV [1, 8, 9] . Thus, there is an enormous unmet need for novel therapeutic strategies to attenuate systemic inflammation and activation of innate immunity in chronic treated HIV. Apolipoprotein A-I (apoA-I) mimetic peptides bind bioactive lipids and endotoxin (LPS) with higher affinity than apoA-I and may be novel therapeutic agents for treatment of inflammatory diseases including cardiovascular and inflammatory bowel disease and cancer [10] [11] [12] [13] [14] . We have shown that an apoA-I mimetic peptide called 4F improved ex vivo antioxidant/antiinflammatory activities of HDL from HIV-1 infected individuals with suppressed viremia on potent ART [15] . 4F attenuates gut inflammation in murine models of gut inflammation when given orally [14] . Importantly, 4F has been tested in humans [16, 17] and has a safety profile that would favor its clinical testing in HIV. Another peptide named 6F that is expressed as a transgene in tomatoes, when concentrated (Tg6F) and given orally, attenuates cancer, cardiovascular and inflammatory bowel disease in mice [11] [12] [13] [14] . Tg6F acts in the intestine, is not absorbed intact in the blood and attenuates M/M activation and intestinal inflammation [11] [12] [13] [14] . Preclinical studies need to validate the therapeutic use of apoA-I mimetic peptides in inflammation in HIV. Herein, we tested at the preclinical level whether the apoA-I mimetic peptides 6F and 4F may attenuate intestinal and systemic inflammation in chronic treated HIV. To bypass limitations of preclinical animal models of chronic treated HIV, we recently described a robust translational preclinical approach, where we used both NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) and Graft versus host disease (GVHD)-resistant C57BL/6 recombination activating gene 2 (Rag2)γcCD47 triple knockout (TKO) BLT mice [18] [19] [20] [21] [22] [23] on independent ART regimens and gut explants from HIV infected participants to demonstrate that apoA-I mimetics attenuate innate immune activation, gut barrier dysfunction, plasma and intestinal oxidized lipoproteins in chronic treated HIV [24] . Herein, using murine biospecimens (blood and gut tissues) and human biospecimens (gut tissues) from our recently published study [24] , we demonstrate that apoA-I mimetics consistently reduce plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) and gut protein levels of the chemokine receptor CX3CR1. ApoA-I mimetics also reduced gut protein levels of the A disintegrin and metalloprotease 17 (ADAM17), an inflammation-inducible enzyme that is responsible for the protease-driven shedding of TNF-α, CX3CL1 and sCD163 [25] , one of the most robust biomarkers of innate immune activation that is strongly associated with mortality in chronic treated HIV [9] . We also show that apoA-I mimetics attenuate ex vivo lipopolysaccharide (LPS)-and oxidized lipoprotein-induced upregulation of the sheddase ADAM17 that mediates release of TNF-α and CX3CL1 in gut explants of uninfected and HIV infected participants on potent ART [25] . Collectively our pre-clinical data demonstrate the potential therapeutic use of apoA-I mimetics to target the cross-talk between oxidized lipoproteins, LPS and ADAM17 to attenuate proinflammatory intestinal and systemic responses in chronic treated HIV. HIV infected BLT mice are an established preclinical model to study in vivo novel therapeutic agents for immune responses and inflammation in HIV [18] [19] [20] [21] [22] [23] . We generated two independent (TKO and NSG) BLT models of humanized mice with functional human immune cells [24] that were mock-infected or infected with HIV-1 (HIV + ) and then treated with clinically relevant ART (HIV + ART + ) together with a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (HIV + ART + Tg6F + ) or a concentrate of transgenic control tomatoes ((HIV + ART + EV + ), as described in Materials and Methods. Using a sensitive Luminex immunoassay platform in plasma of BLT mice, we detected major human and murine cytokines [interleukin (IL)-1β, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α)] and chemokines [C-C Motif Chemokine Ligand 2 (CCL2), CCL3, CCL5, C-X3-C Motif Chemokine Ligand 1 (CX3CL1), C-X-C Motif Chemokine Ligand 10 (CXCL10)] in plasma of both GVHD-prone NSG (S1A, S1B, S1C and S1D Fig We then determined the impact of HIV-1 infection and ART on plasma cytokines and chemokines of innate immunity that contribute to systemic inflammation in chronic treated HIV [1, 8, 9] . Both NSG ( Fig 1A) and TKO ( Fig 1B) HIV + ART + BLT mice had increased plasma h-IL-1β, h-IL-6 and h-TNF-α compared to uninfected mice. Both NSG (S3A In both BLT models other human and murine plasma cytokines and chemokines did not increase in HIV + ART + mice compared to uninfected mice (Figs 1A, 1B, 1C, 1D and S3A, S3B, S3C, S3D). Overall, like our prior observations in studies of treated HIV that showed that potent ART does not attenuate inflammation [26] , our data suggest that potent ART that suppressed plasma viremia [24] does not attenuate systemic inflammation in both BLT models of chronic treated HIV. We hypothesized that oral apoA-I mimetic peptides like Tg6F that have anti-inflammatory properties [27] and attenuate proinflammatory microbial products like LPS and bioactive lipids [10, 28, 29] , may attenuate intestinal and systemic inflammation in chronic treated HIV. To test our hypothesis, we started Tg6F treatment after the viremia was suppressed which was four weeks after the start of the experiment [24] . Tg6F was given in chow diet (0.06% by weight) to the HIV + ART + Tg6F + TKO group. The uninfected (HIV -), HIV + and HIV + ART + mice were all fed with chow diet that contained control transgenic tomato concentrate (EV) to ensure that the effectiveness of Tg6F is due to the presence of 6F, which is not present in EV [11, 13] . We also cotreated HIV + ART + NSG BLT mice fed chow diet with 0.06% Tg6F (wt/wt) with systemic ART for 10 weeks. Both HIV + ART + Tg6F + TKO and NSG BLT mice treated with Tg6F therapy for up to 14 weeks had consistently reduced plasma human cytokines (IL-1β, IL-6 , TNF-α) (Fig 1A and 1B) and human chemokines (CCL2, CCL3, CCL5, CX3CL1) ( Fig 1C and 1D ) compared to HIV + ART + mice BLT mice. Both HIV + ART + Tg6F + TKO and NSG BLT mice treated with Tg6F therapy for up to 14 weeks had also consistently reduced plasma murine cytokines (IL-1β Gut inflammation contributes to gut barrier dysfunction and systemic inflammation in chronic treated HIV [1, 8, 9, 30] . Given that apoA-I mimetics act in the small intestine (SI) and the gut to attenuate systemic inflammation in murine models of inflammatory diseases [11] [12] [13] [14] , we further assessed gut inflammation in BLT models of chronic treated HIV. To minimize confounding effect of GVHD on gut inflammation in NSG BLT mice and given limited amount of total human protein in intestinal humanized mouse tissue, we used sensitive multiplex Luminex immunoassays to assess several major human cytokines and chemokines at the SI of GVHD-resistant TKO BLT mice (S2E and S2F Fig) . HIV + ART + TKO BLT mice had increased cytokines (h-IL-8, KC, h-TNF-α and m-TNF-α) (Fig 2A and 2B ) and chemokines (h-CCL2, m-CCL2, h-CCL3, h-CX3CL1, m-CX3CL1) (Fig 2C and 2D ) compared to uninfected TKO BLT mice. Overall, in both blood and intestine, potent ART did not attenuate increased intestinal levels of human and murine CX3CL1 and TNF-α in HIV-infected ARTtreated BLT mice compared to uninfected mice. In the HIV + ART + group, protein levels of m-TNF-α and m-CX3CL1 in gut, that are known instigators of gut barrier dysfunction [31] , were associated with total protein levels of plasma sCD14 and m-IFABP (S4A, S4B, S4C, S4D, S4E, S4F, S4G and S4H Fig) . Thus, increased TNF-α and CX3CL1 in gut and blood were associated with gut barrier dysfunction and inflammation in both BLT models of chronic treated HIV despite potent ART. CX3CL1 was the most abundant human and murine chemokine that was consistently elevated in BLT mouse models of chronic treated HIV and is known to contribute to inflammation [32] , gut barrier function [33] , HIV progression and end organ disease [6, 34, 35] . The major chemokine receptor of CX3CL1 in M/M is CX3CR1 and is known to interact with bioactive lipids [36] and contribute to gut inflammation [37] . To further confirm our data that increased CX3CL1 levels in the gut contributes to gut inflammation in HIV infected BLT mice, we used a density gradient-and collagenase-free method to isolate the relatively less abundant human myeloid cells and flow cytometry to determine protein levels of CX3CR1 in single cell suspension from SI of TKO BLT mice exposed to HIV and ART. Gating strategy for assessment of intestinal cells in TKO BLT mice is shown in S5 Fig. Compared to uninfected mice, HIV + ART + TKO had increased membrane protein expression (MFI) of hCX3CR1 in hCD33 + gut tissue myeloid cells and tended to have increased murine m-CX3CR1 in mCD11b + gut tissue myeloid cells (S6A, S6B and S6C Fig) . Thus, consistent with prior data that increased CX3CL1-CX3CR1 axis contributes to gut inflammation [37] , the CX3CL1-CX3CR1 axis was upregulated in myeloid intestinal cells in HIV infected BLT mice. ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV Given that Tg6F is not systemically absorbed and works in the gut [11, 13, 14] , we hypothesized that Tg6F attenuates both intestinal and systemic inflammation in BLT models of chronic treated HIV. HIV + ART + Tg6F + TKO BLT mice had reduced intestinal human and murine cytokines (IL-1β, IL-6, IL-8, KC, TNF-α) (Fig 2A and 2B) , chemokines (CCL2, CX3CL1) (Fig 2C and 2D ) and h-CCL3 ( Fig 2C) compared to EV-fed HIV + ART + BLT mice. In the HIV + ART + Tg6F + group, total protein levels of m-TNF-α and m-CX3CL1 in SI were not associated with plasma sCD14 and m-IFABP (S4 Fig). Compared to EV-fed HIV + ART + TKO BLT mice, the Tg6F-fed HIV + ART + TKO BLT mice expressed lower levels of CX3CR1 in human and murine myeloid intestinal cells (S6B and S6C Fig) in SI. Thus, confirming our previous reports that Tg6F attenuates intestinal inflammation [11, 13, 14] , Tg6F attenuated several proinflammatory cytokines and chemokines at both the gut and blood level and intestinal CX3CR1 in BLT models of chronic treated HIV. and HIV + ART + /Tg6F + (blue color)(n = 15) mice BLT mice was expressed as fold to the mean value of each measurement in uninfected (n = 8) BLT mice (within the same cohort). A. Fold changes of human cytokines in gut tissue lysates of HIV + ART + and HIV + ART + /Tg6F + TKO mice compared to uninfected BLT mice. B. Fold changes of murine cytokines in gut tissue lysates of HIV + ART + and HIV + ART + / Tg6F + TKO mice compared to uninfected BLT mice. C. Fold changes of human chemokines in gut tissue lysates of HIV + ART + and HIV + ART + /Tg6F + TKO mice compared to uninfected BLT mice. D. Fold changes of murine chemokines in gut tissue lysates of HIV + ART + and HIV + ART + /Tg6F + TKO mice compared to uninfected BLT mice. Data represent box and whiskers with minimum, median and maximum values (n = 8-15 mice per group). Datapoints represent mean of at least 2 experimental replicates per mouse. The Kruskal Wallis was used to compared >2 groups and the Mann-Whitney test was used to compare 2 groups ( � p < 0.05, �� p < 0.01, ��� p < 0.001). The asterisks in blue demonstrate the comparison relative to the uninfected group. https://doi.org/10.1371/journal.ppat.1010160.g002 ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV We have shown both NSG and TKO HIV + ART + BLT mice had increased biomarkers of human M/M activation (h-sCD14, h-sCD163) and gut barrier dysfunction (IFABP) compared to uninfected mice [24] . Notably, most of the measures of systemic inflammation that were consistently elevated in both BLT models of chronic treated HIV at the blood and gut level, are products of the sheddase activity of the protease ADAM17. ADAM17 is present in macrophages and enterocytes (to impact I-FABP) [25, [38] [39] [40] and mediates the release of sCD14, sCD163, TNF-α, IL-6, CX3CL1, IFABP by M/M and/or enterocytes [41] [42] [43] [44] [45] . We therefore hypothesized that the ADAM17 protein is upregulated in gut tissue of HIV + ART + mice. To test our hypothesis, we used flow cytometry to determine protein levels of ADAM17 in single cell suspension from SI of GVHD resistant TKO BLT mice exposed to HIV and ART. Protein levels of ADAM17 were increased in human h-CD33 + h-CD45 + myeloid cells (Fig 3A and 3B ), murine CD11b + CD45 + myeloid immune cells (Fig 3C and 3D) , murine CD326 + CD45epithelial cells (Fig 3E and 3F ) and murine CD31 + CD45endothelial cells (Fig 3G and 3H ) of HIV + ART + TKO BLT mice compared to uninfected mice. Thus, ADAM17 is a key proinflammatory protein that was consistently increased in myeloid, epithelial and endothelial cells in the intestine of BLT mice with chronic treated HIV. Given that apoA-I mimetics bind LPS and reduce formation of oxidized lipoproteins that are inducers of ADAM17 [25, 46] , we then investigated whether Tg6F reduces ADAM17 levels in BLT mouse models of chronic treated HIV. Tg6F decreased cellular protein levels of ADAM17 in human and murine myeloid cells and murine epithelial and endothelial cells of HIV + ART + Tg6F + BLT mice to levels similar to cellular levels of HIV -BLT mice (Fig 3A, 3B , 3C, 3D, 3E, 3F, 3G, 3H). Overall, our data demonstrated that Tg6F attenuated protein levels of ADAM17 in the gut that contributes to increased gut and systemic inflammation, in chronic treated HIV. Both TNF-α and CX3CL1 are major products of ADAM17 sheddase [25] that were consistently elevated in blood and gut of HIV + ART + BLT mice and are known to contribute to inflammation [32] and gut barrier function [33] . Given known limitations of BLT mouse models of chronic treated HIV [24] and that Tg6F is not yet available for clinical trial in humans, we used colon tissues of uninfected (n = 10) and HIV infected (n = 10) ART-treated 50-60 years old white men without clinical morbidity to further validate our results that apoA-I mimetic peptides attenuate increased ADAM17 protein levels and its sheddase activity (protein levels and TNF-α and CX3CL1) in chronic treated HIV. Gut explants of HIV + participants had higher protein levels of ADAM17 in myeloid (Fig 4A and 4B ) and epithelial cells (Fig 4C and 4D ) and higher protein levels of secreted TNF-α ( Fig 4E) and CX3CL1 ( Fig 4F) compared to uninfected participants. 4F tended (0.052 groups and the Mann-Whitney test was used to compare 2 groups ( � p < 0.05, �� p < 0.01, ��� p < 0.001). https://doi.org/10.1371/journal.ppat.1010160.g003 ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV we hypothesized that the inhibitory effect of apoA-I mimetic peptides on ADAM17 is mediated through LPS. Consistent with this hypothesis, increased protein levels of murine ADAM17 in murine epithelial cells of HIV infected BLT mice on potent ART were positively associated with increased protein levels of biomarkers of gut barrier dysfunction (sCD14, I-FABP) and immune activation (sCD14, sCD163) (S1 Table) . Protein cellular levels of ADAM17 did not correlate with plasma h-sCD14, h-sCD163, m-IFABP in HIV + ART + Tg6F + TKO BLT mice with an intervention that is known to bind LPS [14] (S1 Table) . We then tested whether LPS triggers ex vivo protein levels of ADAM17 and secretion of TNF-α and CX3CL1 from colon tissues of uninfected and HIV infected ART-treated persons. Gut explants of HIV + participants treated with LPS had higher protein levels of ADAM17 in myeloid CD33 + CD45 + ( Fig 5A and 5B ) and CD324 + CD45epithelial cells (Fig 5C and 5D ) compared to vehicle controls (Fig 4A, 4B, 4C and 4D ). Gut explants of HIV + participants treated with LPS had also higher protein levels of TNF-α ( Fig 5E) and CX3CL1 (Fig 5F) compared to vehicle controls (Fig 4E and 4F ). These results were not observed in LPS-treated gut explants from uninfected persons (Figs 4B, 4D, 4E, 4F and 5B, 5D, 5E, 5F). Both 4F and 6F reduced ADAM17 in myeloid cells (Fig 5A and 5B ) and epithelial cells (Fig 5C and 5D ) and secretion of TNF-α ( Fig 5E) and ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV CX3CL1 ( Fig 5F) from LPS-treated gut explants from both uninfected and HIV + participants. Overall, our data suggest that apoA-I mimetic peptides attenuate the crosstalk between increased gut barrier dysfunction, LPS, ADAM17 and associated production of TNF-α and CX3CL1 in gut tissue that contribute to gut and systemic inflammation in chronic treated HIV. Oxidized lipoproteins play a major role in inflammation and immune activation in chronic treated HIV and are associated with sCD163, a major product of the ADAM17 sheddase [46] . Altered lipoproteins also trigger ADAM17 activity [50] [51] [52] . We have previously shown that Tg6F attenuates plasma and intestinal oxidized high-and low-density lipoproteins (HDLox and LDLox) in BLT mouse models of chronic treated HIV [24] . Thus, we further explored the associations of intestinal gut lipoproteins with gut protein levels of ADAM17. Murine intestinal protein levels of ADAM17 were positively associated with intestinal HDLox but not ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV LDLox in HIV + ART + TKO BLT mice (S1 Table) . Protein cellular levels of ADAM17 did not correlate with plasma HDLox, in HIV + ART + Tg6F + TKO BLT mice (S1 Table) , consistent with our prior data that Tg6F is an intervention that reduces HDLox [24] . Gut explants of HIV + and uninfected participants treated with HDLox (Fig 6) had higher intestinal protein levels of ADAM17 in myeloid CD33 + CD45 + (Fig 6A and 6B ) and CD324 + CD45epithelial cells (Fig 6C and 6D ) and intestinal TNF-α ( Fig 6E) and CX3CL1 (Fig 6F) compared to vehicle controls (Fig 4A, 4B, 4C, 4D, 4E and 4F ). Gut explants of HIV + and uninfected participants treated with LDLox (Fig 7) had also higher intestinal protein levels of ADAM17 in myeloid CD33 + CD45 + (Fig 7A and 7B ) and CD324 + CD45epithelial cells (Fig 7C and 7D ) and intestinal TNF-α ( Fig 7E) and CX3CL1 (Fig 7F) compared to vehicle controls (Fig 7A, 7B , 7C, 7D, 7E and 7F). Both 4F and 6F reduced ADAM17 in myeloid cells (Fig 6A and 6B ) and epithelial cells (Fig 6C and 6D ) and secretion of CX3CL1 (Fig 6F) from HDLox-treated gut explants from both uninfected and HIV + participants. 4F tended (p = 0.089) to reduce secretion of TNF-α from HDLox-treated gut explants from uninfected participants (Fig 6E) . 4F and 6F reduced secretion of TNF-α from HDLox-treated gut explants from HIV + participants ( Fig Fig 6. Ex vivo ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV 6E). 4F and 6F also reduced ADAM17 in myeloid cells (Fig 7A and 7B ) and epithelial cells ( Fig 7C and 7D) and secretion of TNF-α ( Fig 7E) and CX3CL1 (Fig 7F) from LDLox-treated gut explants from HIV + participants. 4F also reduced ADAM17 in myeloid cells (Fig 7A and 7B ) and epithelial cells (Fig 7C and 7D ) from LDLox-treated gut explants from uninfected participants. 4F and 6F consistently and similarly reduced protein levels of ADAM17 in both myeloid and epithelial cells in gut explants from HIV + participants with or without LPS (Fig 5) , HDLox (Fig 6) and LDLox (Fig 7) treatments. Overall, our complementary BLT mouse and ex vivo human studies, demonstrated that apoA-I mimetic peptides attenuate proinflammatory effects of LPS and oxidized lipoproteins on ADAM17 and its sheddase activity that promote immune activation, systemic and intestinal inflammation in chronic treated HIV (Fig 8) . In independent preclinical models of chronic treated HIV, including BLT mice and human gut explants, we show that apoA-I mimetics consistently attenuated increased mediators of intestinal and systemic inflammation that are products of the ADAM17 sheddase activity (TNF-α, CX3CL1). ApoA-I mimetics also attenuated protein levels of ADAM17, that reflect signaling of both LPS [47] [48] [49] and bioactive lipids [50] [51] [52] in myeloid and intestinal cells and drive shedding of biomarkers of inflammation and morbidity such as TNF-α, IL-6, CX3CL1, sCD14, sCD163 and I-FABP [1, 8, 9, 30] in chronic treated HIV. We also demonstrate that apoA-I mimetic peptides through favorable effects on bioactive lipids, dysfunctional HDL and LPS, that disrupt lipid rafts [53, 54] and activate CX3CR1 and ADAM17 [50] [51] [52] , alters cytokines and immune cells in the intestinal wall. While low-level of HIV-1 replication may be seen in the BLT models, we previously demonstrated that Tg6F did not have an impact on viral replication [24] and the impact on biomarkers of inflammation was due to effect of Tg6F on LPS and oxidized lipoproteins. The component of the oral ApoA-I mimetic peptide Tg6F accounting for the beneficial effects on systemic inflammation is the 6F peptide, since control [11, 13] had no effect on biomarkers of inflammation in chronic treated HIV. Given that Tg6F seems to work exclusively in the gut [11] [12] [13] [14] , our model (Fig 8) is proof-of-concept that therapeutic targeting of intestinal inflammation, intestinal LPS and bioactive lipids and gut barrier dysfunction may have a favorable impact on inflammation and immune activation that can complement ART to attenuate development of comorbidities like cardiovascular disease in chronic treated HIV. Chronic treated HIV is a state of increased levels of bacterial translocation, and LPS signaling as evidenced by high levels of sCD14 [1, 8, 9] . Activation of the TLR pathway by LPS is dependent on the co-localization of TLR4 and CD14 in lipid rafts that permit the activation of downstream signaling that culminate in NF-κB activation and the synthesis of pro-inflammatory cytokines [55] . Notably, ADAM17 is implicated in shedding of TLR4 [47] . LPS has also been shown to directly upregulate in vitro ADAM17 levels in immune, epithelial and endothelial cells [48, 49] . ADAM17 mediates further release of TNF-α that disrupts epithelial cell tight junctions [31] and triggers release of I-FABP [56] . In HIV + ART + BLT mice protein levels of ADAM17 and plasma TNF-α and CX3CL1 positively associated with sCD14, suggesting a link between LPS signaling and ADAM17 protein levels in our BLT models of chronic treated HIV. Notably, in both BLT models, secretion of proinflammatory cytokines (TNF-α, IL-6) and chemokines (CX3CL1) and protein levels of ADAM17 were also increased in murine cells that are not infected by HIV suggesting a direct proinflammatory effect of murine LPS that was upregulated in BLT models of chronic treated HIV, independently of different ART regimens. These data are consistent with prior evidence that elevated LPS levels [2, 8, 46] are associated with biomarkers of increased morbidity in chronic HIV infection and trigger ADAM17 sheddase activity [25] . We also provide evidence based on gut explants of HIV-1 infected persons that apoA-I mimetics reduce LPS-induced expression of ADAM17 in intestinal epithelial and myeloid cells. These data are consistent with our prior findings that apoA-I mimetics attenuate chronic immune activation through reducing biomarkers of gut barrier dysfunction and bacterial translocation in preclinical models of chronic treated HIV [24] . Tg6F also reduced inflammation and small intestine enterocyte and plasma levels of LBP, CD14, TLR4 and MyD88 [57] in murine models of disease [57, 58] . Overall, our data are consistent with prior human studies that increased LPS and bacterial translocation are associated with systemic inflammation and immune activation in chronic treated HIV [1] . ART treated HIV infected individuals may have higher oxidative stress compared to HIV infected naïve or healthy subjects due to alterations in antioxidant systems [59, 60] . During oxidative stress oxidized phospholipids acquire pleomorphic proinflammatory effects [61] [62] [63] [64] [65] . In vivo mechanistic animal models demonstrated that oxidized lipids are involved in the pathogenesis of inflammation and can be targeted therapeutically [66] . Several bioactive lipids interact with CX3CR1 [36, 67] that through NF-κB and CX3CL1 signaling contribute to inflammation and gut barrier function [6, [32] [33] [34] [35] 44, 45, 68] . Bioactive lipids may also disrupt epithelial cell tight junctions either directly [69] or through lipid raft disruption [53, 54] or TNF-α signaling [31] that collectively promote gut barrier dysfunction, intestinal inflammation, bacterial translocation and ultimately systemic inflammation. Consistent with our data in humans [46] , plasma and intestinal HDLox levels rather than LDLox were associated with sCD163 [24] and ADAM17 in HIV + ART + BLT mice. While HDL is generally an anti-inflammatory lipoprotein during systemic inflammation it can be oxidized (HDLox) and becomes dysfunctional [15, [70] [71] [72] . Our data are consistent with data from others that dysfunctional HDL and altered lipoproteins disrupt lipid rafts [54] , contribute to increased protein levels of ADAM17 [50] and activate ADAM17 [50] [51] [52] . Oxidized LDL also carries oxidized lipids and like HDLox may also contribute to inflammation in HIV [46, 73] . Notably, the associations between plasma and gut bioactive lipids with biomarkers of gut and systemic inflammation in the HIV + ART + EV + group were attenuated in the HIV + ART + Tg6F + group with an intervention that directly attenuates oxidized lipoproteins in vivo [24, 58] . We confirmed these observations in preclinical ex vivo human gut explant models where both 4F and 6F directly attenuated ADAM17 and inflammatory responses triggered by oxidized lipoproteins. Importantly, in all models we consistently found that apoA-I mimetics attenuated all major innate immunity biomarkers of inflammation that predict morbidity in chronic treated HIV including IL-1β, IL-6, TNF-α, CX3CL1, sCD14 and sCD163 [1] [2] [3] [4] [5] [6] 8, 9, 24, 25] . In addition, both IL-1β [74] and LPS that are also associated with increased morbidity in chronic HIV infection [2, 8] trigger ADAM17 sheddase activity [25] . Although ADAM17 levels have not been determined in chronic treated HIV, high levels of sCD163, a product of increased sheddase activity of ADAM17, in HIV infected ART-treated persons [1] [2] [3] [4] [5] [6] 8, 9] and our preclinical data in HIV infected BLT mice, suggest increased protein levels of ADAM17 in treated HIV. Our data are consistent with previous evidence that ADAM17 is a potential therapeutic target for several inflammatory conditions [75] [76] [77] including viral infections (such as COVID-19) [78] . We propose the first therapeutic strategy that can target ADAM17 activity in chronic treated HIV. Collectively, prior evidence [46] [47] [48] [49] [50] [51] [52] 73] and our data suggest that increased LPS and oxidized lipoproteins drive increased ADAM17 activity as inducer of gut and systemic inflammation in chronic treated HIV. Importantly, in a prior clinical trial of 4F in humans, 4F was given parenterally and not orally [17] and did not improve HDL functionality [17] . Further work from our group demonstrated that apoA-I mimetics like 4F and 6F work primarily in the gut to inhibit gut inflammation and M/M activation [11] [12] [13] [14] , and oral apoA-I mimetics demonstrate therapeutic efficacy in several inflammatory diseases in animal models including cancer, cardiovascular and inflammatory bowel disease [11] [12] [13] [14] . The safety and pharmacokinetics of oral apoA-I mimetic peptides have been tested in humans [16] and mice [11] [12] [13] [14] . ApoA-I mimetic peptides seem to achieve higher concentration in the proximal small intestine [11] [12] [13] [14] . This may explain how Tg6F that is not systemically absorbed [11, 13, 14] , had a major favorable impact on systemic inflammation and immune activation in BLT models of chronic treated HIV. Our study has limitations. Humanized mice do not fully recapitulate all aspects of HIV-1 immunopathogenesis due to presence of GVHD, suboptimal variable engraftment of human lymphocytes and macrophages, absence of fully functional secondary human lymphoid tissues and non-human microbiome [18] [19] [20] [21] [22] [23] . Gut explants also only examine early effects of signaling. Thus, our findings may be explained by other underlying mechanisms or variables that were not studied in our preclinical models. Despite the limitations of humanized mouse models, we have shown the capacity of independent BLT mouse models to support HIV infection, express clinically relevant biomarkers of intestinal and systemic inflammation in the setting of chronic treated HIV and consistently respond to clinically relevant ART [24] . We excluded NSG mice with clinical GVHD and we also included studies with GVHD-resistant TKO BLT mice [21] [22] [23] . Gut explants also do not have limitations of other ex vivo intestinal models [79] and ex vivo addition of 4F in gut explants is the only preclinical approach in humans at this time. We did not include gut explants from HIV-1 infected women. Unlike other studies [80] , we did not study the differential impact of hormones and sex-driven differences on the regulation of the gut inflammatory milieu in the setting of ex vivo use of apoA-I mimetics. We also did not study the differential impact of HIV-1 per se, antivirals, age, ethnicity and comorbidities on the regulation of the gut inflammatory milieu in the setting of ex vivo use of apoA-I mimetics. Further human studies are needed to determine the impact of apoA-I mimetic peptides on gut inflammation among different patient groups based on age, gender, ethnicity and comorbidities. Overall, our robust experimental approach with independent preclinical in vivo and ex vivo experimental models and consistent findings provides important preclinical insight into the therapeutic impact of apoA-I mimetics on inflammation in chronic treated HIV. Our data support the hypothesis that apoA-I mimetic peptides attenuate a cycle of production of bioactive lipids and LPS that contribute to increased intestinal and systemic inflammation in chronic treated HIV. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (" The Guide"), and was approved by the Institutional Animal Care and Use Committees of the University of California, Los Angeles, protocol # 1997-176. All individuals enrolled in the study provided written informed consent and the study was approved by the University of California Los Angeles Institutional Review Board [24] . A concentrate of control transgenic tomatoes (EV) or a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) were prepared and added to the diets at 0.06% by weight as described previously [11] . 4F and 6F peptides were synthesized as previously [14] . The antivirals Emtricitabine (FTC), Tenofovir Disoproxil Fumarate (TDF), Raltegravir (RAL), abacavir (ABC), dolutegravir (DOL), lamivudine (3TC), tenofovir alafenamide (TAF), FTC were either a generous gift from Gilead Sciences or were obtained from pharmacy. HIV p89.6 plasmid DNA was obtained from the NIH AIDS reagent Program. Murine chow diet with antibiotic was purchased from Envigo Teklad Diets. All other materials were purchased from commercially available sources and have been previously described in detail [24] . The current study used murine biospecimens (blood and gut tissues) from a previously published study [24] to generate the data in this manuscript. As previously described [24] animal protocols were carried out in accordance with all federal, state, and local approved guidelines. The TKO or NSG BLT mice were generated and maintained as described [24] . A total of 5 (2 TKO and 3 NSG) independent cohorts of mice (each constructed from the same human donor tissues) were used for various experiments and were pooled for comparing mice that were uninfected (Group A: HIV -), infected and on potent ART (group B: HIV + , antivirals) and infected on potent ART and Tg6F (group C: HIV + , antivirals, Tg6F)(n = 10-20 mice per group) [24] . Given the variable engraftment of human immune cells and to better compare results among mice, we determined changes in human cytokines and chemokines among mice groups over 16 weeks of potent ART and we compared them to the uninfected group within each cohort (identical human donor) [24] . All the data from independent cohorts were then pooled together [24] . In the TKO BLT mice, Tg6F was given the day after the viral load was found to be suppressed while in the NSG BLT mice it was initiated 2 weeks before confirmation of suppression of viremia [24] . Between 16 and 18 weeks, mice were challenged intraperitoneally with 500 ng p24 of HIV-1 dual-tropic 89.6 virus as described previously [24] . As previously described [24] , TKO C57 Bl6 mice were treated with ART consisted of combination medication of ABC/DOL/3TC (Triumeq) resuspended in the sweetened water gel formulation (Sucralose) as previously described [81] . As previously described [24] , NSG BLT mice were treated with ART regimen consisting of TDF (8.75 mg/kg)/FTC (13 mg/kg)/RAL (17.5 mg/kg). ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV The current study used human biospecimens (gut tissues) from a previously published study [24] . HIV-seronegative (n = 10) and seropositive (n = 10) 50-60 years old participants were recruited in the Gastroenterology Unit of UCLA as previously described [24] . To avoid confounding effects from sex, race, inflammation (other than HIV) on M/M activation, we selected HIV + white men, with no known clinical disease other than HIV or risk factors for clinical disease (e.g., metabolic syndrome, diabetes, dyslipidemia, use of lipid lowering medication). Six participants were on elvitegravir, cobicistat, emtricitabine (FTC), tenofovir alafenamide (TAF) and four participants were on bictegravir/FTC/TAF. All HIV infected persons had suppressed viremia (HIV-1 RNA <50 copies/ml), CD4 + T cells > 500 cells/mm 3 , duration of ART therapy between 3.2-6.5 years and nadir CD4 + T cell count > 250 cells/mm 3 . Ten healthy white men were also included as previously described [24] . Biopsy specimens were obtained endoscopically from the rectosigmoid colon and gut mucosal explants were processed as previously described [24] . Briefly, 10-15 biopsies per participant were collected and cultured using RPMI 1640 medium with amphotericin B and piperacillintazobactam on absorbable gelatin sponge in tissue culture plates in a 37˚C humidified incubator for 72 hours [24] . Triplicate biopsies were treated with media (control) or oxidized lipoproteins (HDLox, LDLox) at concentration 25 μg/ml [71, 82] and/or with LPS at concentration 100 μg/ml [83] and/or with 4F, 6F at concentration 100 μg/ml [14] for 72 hours as previously described [14, 71, 82, 83] . Supernatants were collected to measure secreted protein levels of cytokines (TNF-α) and chemokines (CX3CL1), that are mediated by ADAM17 sheddase activity. As previously described [24] , the epithelial cells were isolated using DTT and EDTA while the remaining intestinal wall was further digested with the enzyme collagenase IV as previously described [84] . Protein levels of ADAM17 were determined in myeloid CD33 + CD45 + and CD324 + CD45epithelial cells of gut explants by flow cytometry. LDL was isolated from plasma of healthy subjects and the lipoproteins were oxidized by use of copper ions as previously [71, 82] . HDL isolated from healthy participants was oxidized in vitro with 13(S)-HPODE as described [85] . Briefly, human HDL (5 μg HDL-cholesterol/ml) was incubated with 13(S)-HPODE (0.5 μg/ml), for 60 min (co-incubation). 13(S)-HPODE oxidized HDL and impaired HDL function in wild type C57BL/6J mice [85] . Thus, in vitro oxidation of HDL with 13(S)-HPODE is more physiologically relevant compared to copper ions that do not contribute to HDL dysfunction and oxidation of HDL in vivo [85] . For studies involving Tg6F, as previously described [24] , the final diets contained 0.06% of Tg6F extract by weight and were administered for 12 weeks as previously described [11, 12] . Triplicate biopsies were treated with 4F, 6F or sham peptide at concentration 100 μg/ml for 72 hours as previously described [14] . 50-100 mg of gut tissue samples were placed in 2 ml screwcap dissociation tubes with ceramic beads (Precellys) and were mechanically dissociated in T-PER tissue protein extraction Reagent (Thermo Scientific) at power 5000 with two 20-second cycles as previously described ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV [24] . Plasma and gut tissue human and murine cytokines (IL-1 beta, IL-6, h-IL-8, m-KC, IL-10, TNF-alpha), chemokines (CCL2, CCL3, CCL5, CX3CL1, CXCL10, h-IL-8/CXCL8) were determined using the human and murine magnetic Luminex performance assay kits according to the manufacturer instructions (R&D) and as previously described [24] . Murine CX3CL1/ Fractalkine was determined by Quantikine ELISA as per manufacturer (R&D). Statistics P values less than 0.05 by Kruskal-Wallis or Mann-Whitney were considered significant. For all correlations, Spearman's correlation coefficient was calculated. In the setting of exploratory approach we did not adjust for multiple comparisons since commonly-used multiple testing adjustment methods assume independence of tests, which in protein expression studies and in our explored pathways translates to a questionable assumption that all explored measures operate independently [86] . Instead, consistency between 2 independent BLT models, direction, and magnitude of the correlation coefficient in conjunction with the nominal p values were considered in order to help distinguish true and false-positive findings. All analyses were performed with Graphpad, version 7.0. Supporting information S1 Dataset. Includes the raw data used to make Figs 1A, 1B, 1C, 1d, 2A, 2B, 2C, 3B, 3D, 3F, 3H, 4B, 4D, 4E, 4F, 5B, 5D, 5E, 5F, 6B, 6D, 6E, 6F, 7B, 7D, 7E, 7F , S1A, S1B, S1C, S1D, S2A, S2B, S2C, S2D, S2E, S3A, S3B, S3C, S3D, S4, S5, S6B, S6C. The mean value of each measurement in HIV + ART + (red color) and HIV + ART + /Tg6F + (blue color) mice BLT mice was expressed as fold to the mean value of each measurement in uninfected BLT mice (within the same cohort). A. Fold changes of murine cytokines in plasma of HIV + ART + (n = 19) and HIV + ART + /Tg6F + (n = 21) NSG mice compared to uninfected (n = 11) BLT mice. B. Fold changes of murine cytokines in plasma of HIV + ART + (n = 14) and HIV + ART + /Tg6F + (n = 15) TKO mice compared to uninfected (n = 8) BLT mice. C. Fold changes of murine chemokines in plasma of HIV + ART + (n = 19) and HIV + ART + /Tg6F + (n = 21) NSG mice compared to uninfected (n = 11) BLT mice. D. Fold changes of murine chemokines in plasma of HIV + ART + (n = 14) and HIV + ART + /Tg6F + (n = 15) TKO mice compared to uninfected (n = 8) BLT mice. Data represent box and whiskers with minimum, median and maximum values (n = 8-21 mice per group). Datapoints represent mean of at least 2 experimental replicates per mouse. The Kruskal Wallis was used to compared >2 groups and the Mann-Whitney test was used to compare 2 groups ( � p < 0.05, �� p < 0.01, ��� p < 0.001). The asterisks in blue demonstrate the comparison relative to the uninfected group. A. Levels (pg/ml) of murine cytokines [interleukin (IL)-1β, IL-6, KC/murine IL8 homolog, IL-10, tumor necrosis factor alpha (TNF-α)] in plasma from uninfected NSG BLT mice after 16 weeks of HIV infection in the HIV + ART + group. B. Fold changes of murine cytokines in plasma of HIV + ART + (red color) and HIV + ART + /Tg6F + (blue color) NSG mice compared to uninfected BLT mice. The mean value of each measurement was expressed as fold to the mean value of each measurement in uninfected BLT mice (within the same cohort). C. Levels (pg/ml) of murine cytokines in plasma from uninfected TKO BLT mice after 16 weeks of HIV infection in the HIV + ART + group. D. Fold changes of murine cytokines in plasma of HIV + ART + (red color) and HIV + ART + /Tg6F + (blue color) TKO mice compared to uninfected BLT mice. The mean value of each measurement was expressed as fold to the mean value of each measurement in uninfected BLT mice (within the same cohort). Data represent box and whiskers with minimum, median and maximum values (n = 8-22 mice per group). Datapoints represent mean of at least 2 experimental replicates per mouse. The Kruskal Wallis was used to compared >2 groups and the Mann-Whitney test was used to compare 2 groups ( � p < 0.05, �� p < 0.01, ��� p < 0.001). The asterisks in blue demonstrate the comparison relative to the uninfected group. 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The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 89.6 Virus from Dr. Ronald Collman. The flow cytometry machine used in the study was purchased through the UCLA Center for AIDS Research (P30AI28697) grant. Writing -review & editing: Maria Daskou, Scott G. Kitchen, Alan M. Fogelman, Srinivasa T.Reddy, Theodoros Kelesidis. ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV