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Ixolaris Reduced Inflammation/Immune Activation,
Prevented HIV Rapid Progression
 
 
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in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation......Ixolaris is a small molecule isolated from the saliva of the tickIxodes scapularis.......Together, these results suggest that Ixolaris treatment had a beneficial effect in reducing immune activation and inflammation associated with SIV infection......Ixolaris administration significantly reduced plasma D-dimer levels in acute SIVsab infection of PTMs -elevated D-dimer, which is associated with increased incidence of cardiovascular comorbidities and mortality.....we next compared the D-dimer levels in Ixolaris-treated PTMs and controls. Ixolaris administration significantly reduced plasma D-dimer levels in acute SIVsab infection of PTMs (P = 0.033) (Fig. 6J) and resulted in lower D-dimer levels during early chronic infection, indicating a clear effect of Ixolaris in improving the coagulation status in the SIV-infected animals. To monitor the infection in SIVsab-infected PTMs, we measured plasma SIV viremia in all infected animals (47). The Ixolaris-treated group showed overall lower viral loads compared to controls (Fig. 6K).......Ixolaris-treated animals did not develop disease during the first 100 days after infection (Fig. 6L). In accordance with our previous studies in which two of five SIVsab-infected PTMs were rapid progressors (48, 49), one of three animals progressed to AIDS in the first 100 days after infection in the untreated group; however, no rapid progression was registered in the Ixolaris group. Statistical significance was not reached in the present study, possibly due to the small sample size. This result is of potential translational interest because it suggests that Ixolaris treatment may abrogate rapid progression in treated animals, likely due to the combined effect of reduced immune activation and inflammation, reduced hypercoagulable status, and small reduction in plasma viremia.
 
These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.TF expression is associated with increased levels of D-dimer (18) and thus may be associated with an increased risk for cardiovascular complications in HIV-infected individuals (19). These findings support a direct role of activated monocytes in the persistent inflammatory milieu observed in chronic HIV infection.....The animals treated with Ixolaris showed significantly reduced levels of the proinflammatory cytokine IL-17 during early chronic infection (P = 0.03; Fig. 6C). The anticoagulant treatment also affected T cell immune activation, as demonstrated by a lower frequency of CD4+ T cells expressing HLA-DR+ and CD38+ (Fig. 6D), and by significantly lower frequency of CD8+ T cells expressing HLA-DR+ and CD38+ during early chronic infection (P < 0.001; Fig. 6E).....After treatment, TF expression on circulating CD14+ monocytes decreased compared with untreated controls (Fig. 6F). In addition, Ixolaris treatment significantly lowered both CD80 expression in chronically infected PTMs (P = 0.004; Fig. 6G) and CD86 expression in both acutely and chronically infected PTMs (P = 0.03; Fig. 6H) compared with the untreated controls.
 
.......These primary observations indicated an increase in the capacity of monocytes to produce TF and promote factor Xa formation in vitro upon cellular activation in HIV infection, which persists after ART-induced HIV suppression......factors driving TF expression by monocytes persist after ART implementation.....Serum treated with this bactericidal compound resulted in the lowest TF expression among all the experimental conditions tested (Fig. 3C). These findings demonstrate that circulating microbial products are relevant drivers of TF expression by activated monocytes....Chronic HIV infection has been associated with persistent immune activation and elevated markers of coagulation (22,32). We hypothesized that TF-expressing monocytes, aside from promoting coagulation, could contribute to systemic inflammation. In non-HIV-infected healthy individuals, TFposmonocytes more frequently produced multiple proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, simultaneously upon LPS stimulation.....Ixolaris is a small molecule isolated from the saliva of the tick Ixodes scapularis and acts as a potent TF pathway inhibitor, by blocking factor VIIa/TF-induced coagulation (38, 39). Although Ixolaris has been tested in thrombosis models (40), it has not been evaluated in the setting of HIV-driven coagulopathy......These results demonstrate that, at low doses, Ixolaris can potently inhibit TF functional activity in cells from HIV+ patients and from SIVsab-infected NHPs and suggest that inhibition of TF activity could be used to suppress monocyte-driven activation of coagulation in these settings without directly affecting the capacity of these cells to respond to TLR stimulation.

1208171

NIAID Scientists Illuminate Mechanism of Increased Cardiovascular Risks with HIV - reduced immune activation
-......https://www.niaid.nih.gov/news-events/niaid-scientists-illuminate-mechanism-increased-cardiovascular-risks-hiv
 

1208172

People living with HIV are up to twice as likely to experience heart attacks, strokes and other forms of cardiovascular disease as people who do not have the virus, even when HIV infection is well-controlled with the use of antiretroviral therapy. Compared with previous data on SIV-infected monkeys, Ixolaris-treated monkeys had lower levels of biomarkers that predict abnormal blood clotting and immune activation. These findings indicate that targeting the TF pathway may reduce some cardiovascular risk factors. While Ixolaris has not been tested for safety or its ability to prevent abnormal clotting in humans, these data suggest that targeting this cellular mechanism may slow the inflammation and clotting processes that place people living with HIV at a higher risk of cardiovascular problems.
 
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Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy Science Translational Medicine 30 Aug 2017
 
Melissa E. Schechter,1* Bruno B. Andrade,2,3,4,5,6* Tianyu He,7,8* George Haret Richter,7Kevin W. Tosh,2 Benjamin B. Policicchio,7,9 Amrit Singh,10 Kevin D. Raehtz,7,11 Virginia Sheikh,10Dongying Ma,12 Egidio Brocca-Cofano,7,8 Cristian Apetrei,7,11 Russel Tracy,13 Ruy M. Ribeiro,14,15Alan Sher,2 Ivo M. B. Francischetti,12 Ivona Pandrea,7,8 Irini Sereti10
 
Abstract
 
In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. A better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidence of robust production of multiple proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)-related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.
 
INTRODUCTION

 
Monocytes are key mediators of innate immunity and have been closely associated with pathogenesis of chronic viral infections, including HIV (1, 2). Heightened circulating levels of monocyte activation markers, such as soluble tissue factor (TF), soluble CD14 (sCD14), and soluble CD163 (sCD163), have been associated with increased risk for death (3), noninfectious complications (4, 5), subclinical atherosclerosis (6), and immune reconstitution inflammatory syndrome (IRIS) in HIV-infected individuals (7). Moreover, differential activation of monocyte subsets has recently been described as a predictor of tuberculosis (TB)-associated IRIS in patients with HIV-TB co-infection (7). One important feature of monocytes in HIV pathogenesis is their capacity to produce TF (8-10). TF is expressed in response to inflammatory stimuli, such as Toll-like receptors (TLRs) (11-13) and cytokine-driven signals (14, 15), and initiates the extrinsic coagulation cascade by cleaving coagulation factors, leading to the formation of factor Xa, thrombin, and fibrin, which, when degraded, forms the coagulation biomarker D-dimer (16, 17). For these reasons, augmented TF expression is associated with increased levels of D-dimer (18) and thus may be associated with an increased risk for cardiovascular complications in HIV-infected individuals (19). These findings support a direct role of activated monocytes in the persistent inflammatory milieu observed in chronic HIV infection.
 
The need to investigate the link between coagulation and inflammation in chronic viral infections is pressing. Inflammatory and coagulation markers are both independent predictors of morbidity and mortality in treated HIV individuals (20-23) and are associated with noninfectious complications of HIV, such as cardiovascular and thromboembolic disease (19), which are rising due to the aging of treated HIV-infected persons (24). In an experimental model of nonhuman primates (NHPs) infected with SIVsab, we previously demonstrated that increases in D-dimer and monocyte activation markers (sCD14) predict disease progression (25). These findings highlighted monocyte activation as a key event driving persistent coagulation in simian immunodeficiency virus (SIV)/HIV chronic infection, suggesting a need to delineate the role of monocyte-derived TF in SIV/HIV-driven systemic inflammation and coagulopathy.
 
Here, we evaluated the role of TF-expressing monocytes in HIV and SIV pathogenesis and related coagulopathy. We examined the links between inflammation and coagulation with the aim to identify potential targets for therapeutic interventions in HIV-infected persons. TF-expressing monocytes are expanded in chronic HIV infection independent of antiretroviral treatment
 
It has been previously observed that monocytes from HIV+ patients display higher levels of TF expression compared to healthy controls (18). Here, we extended these observations by comparing TF expression in HIV+ individuals before and after antiretroviral therapy (ART) initiation (after virological suppression was achieved) and age- and gender-matched healthy controls (table S2). In a cross-sectional comparison of ex vivo TF expression, measured by flow cytometry, we observed that the frequency of TFpos monocytes was significantly higher in both ART-naïve and ART-treated HIV+ patients compared to healthy controls (P < 0.001 and P < 0.01, respectively; Fig. 2A), albeit with considerable variability in expression levels. The median frequency of TFpos cells was not statistically different between treatment-naïve HIV+ patients and those with ART-induced suppression of HIV viremia (Fig. 2A). This finding suggests that TF protein expression in HIV+patients may not be substantially affected by ART. We next compared the potential of monocytes to produce TF in response to LPS between the different study groups. We found that the frequencies of TFpos monocytes were significantly higher in HIV+ patients, before and after ART, compared to healthy individuals in unstimulated cultures as well as upon LPS stimulation (Fig. 2B). There was no observed difference in response to LPS between treatment-naïve HIV+ patients and those on ART with virological suppression (Fig. 2B). These results mirrored the findings obtained by quantification of TF protein expression in PBMC lysates (Fig. 2C). We further demonstrated that TF expressed on the cell surface of monocytes from HIV+ individuals was able to trigger factor Xa formation, demonstrating that TF was functionally active in vitro (Fig. 2D). Again, no difference was detected in TF functional activity between the groups of HIV+ patients (Fig. 2, C and D). The differences in TF expression on monocytes were independent of the total monocyte counts in PBMC among the HIV-infected groups, which were not significantly different (median, 4451 cells/μl; IQR, 2383 to 6829 in ART-naïve versus 4507 cells/μl; IQR, 3308 to 7771 in virologically suppressed individuals; P = 0.34).
 
TF-expressing monocytes produce multiple proinflammatory cytokines
 
Chronic HIV infection has been associated with persistent immune activation and elevated markers of coagulation (22, 32). We hypothesized that TF-expressing monocytes, aside from promoting coagulation, could contribute to systemic inflammation. In non-HIV-infected healthy individuals, TFpos monocytes more frequently produced multiple proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, simultaneously upon LPS stimulation (Fig. 4, A to C). We further investigated the profile of intracellular cytokine expression in monocytes ex vivo from our longitudinal cohort of HIV+ patients before ART initiation and after virological suppression (table S3). HIV+ individuals, regardless of treatment status, exhibited high frequencies of monocytes spontaneously producing proinflammatory cytokines (fig. S6A). Furthermore, by testing the monocyte response to LPS stimulation in our prospective cohort of HIV+ patients, we detected markedly different intracellular cytokine expression profiles between TFneg and TFpos monocytes in ART-naïve HIV+ patients (χ2, P < 0.001). In contrast to monocytes from healthy controls (Fig. 4C), after LPS stimulation, the vast majority of TFneg monocytes from HIV-infected individuals produced IL-6 alone (median, 48.7%; IQR, 33.5 to 52.8% of all the cytokine-producing TFneg cells), whereas TFpos monocytes more frequently produced TNF-α, IL-1β, and IL-6 simultaneously (53.1% of all the cytokine-producing TFpos cells; IQR, 49.5 to 56.7%) (Fig. 4D). The intracellular cytokine expression profile of LPS-stimulated monocytes significantly changed after ART initiation (χ2, P = 0.015 for the expression profile in TFneg cells and P < 0.001 for TFpos monocytes from pre-ART versus post-ART initiation; Fig. 4D). In virologically suppressed HIV+ individuals, TFneg cells exhibited a mixed cytokine expression profile with monocytes producing one or various combinations of two cytokines, whereas most of the TFpos monocytes remained polyfunctional, producing a combination of three cytokines (TNF-α, IL-1β, and IL-6) simultaneously (χ2, P < 0.001 profile of TFneg versus TFpos cells; Fig. 4D). In addition, the frequency of TF-expressing monocytes strongly correlated with that of polyfunctional monocytes before ART initiation (r = 0.89, P < 0.001) and in patients with virological suppression (r = 0.91, P < 0.001).
 
Ixolaris blocks TF activity without suppressing monocyte immune function in vitro The results at this point support the hypothesis that monocytes are highly responsive to thrombin and act as a critical link between TLR-driven persistent inflammation and coagulation in chronic viral infections. Interventional therapies focusing on TF inhibition and/or blockade could therefore be key to breaking this vicious cycle of coagulation and inflammation in HIV and SIV pathogenesis. Ixolaris is a small molecule isolated from the saliva of the tick Ixodes scapularis and acts as a potent TF pathway inhibitor, by blocking factor VIIa/TF-induced coagulation (38, 39). Although Ixolaris has been tested in thrombosis models (40), it has not been evaluated in the setting of HIV-driven coagulopathy.
 
In our in vitro model, Ixolaris inhibited TF functional activity in monocytes from healthy donors stimulated with LPS in a dose-dependent fashion (Fig. 5A). Even at doses 10 times higher than the maximum inhibitory concentration (10 ng/ml), Ixolaris did not exhibit substantial cytotoxicity (fig. S8). We next examined whether Ixolaris blocks TF activity in pathological settings such as HIV and SIV infection. Ixolaris completely inhibited the formation of factor Xa under unstimulated conditions and after LPS stimulation in vitro in PBMC from both ART-naïve and ART-treated, virologically suppressed HIV+ persons (Fig. 5B). Ixolaris also significantly inhibited TF activity in PBMC from chronically SIVsab-infected PTMs and AGMs (Fig. 5C). Ixolaris did not affect TF protein expression or cytokine production triggered by LPS stimulation in vitro (Fig. 5D). These results demonstrate that, at low doses, Ixolaris can potently inhibit TF functional activity in cells from HIV+ patients and from SIVsab-infected NHPs and suggest that inhibition of TF activity could be used to suppress monocyte-driven activation of coagulation in these settings without directly affecting the capacity of these cells to respond to TLR stimulation.
 
In vivo administration of Ixolaris is safe and decreases coagulation and immune activation in SIVsab infection in PTMs
 
To assess the efficacy of Ixolaris, we first measured its anticoagulant effect in vitro (Fig. 6). Ixolaris resulted in dose-dependent prolongation of prothrombin time (PT)/international normalized ratio (INR) when added to human plasma collected from healthy individuals (Fig. 6A). Changes in PT/INR were similar when Ixolaris was added to plasma from uninfected healthy PTMs (Fig. 6B). Activated partial thromboplastin time (aPTT), an intrinsic pathway coagulation marker, was not affected by Ixolaris in either monkey or human plasma, indicating that Ixolaris has a specific inhibitory effect on the extrinsic pathway. Our results thus confirmed the high efficacy and specificity of Ixolaris in NHPs in vitro.
 
Chronic progressive HIV/SIV infection is characterized by high levels of immune activation, inflammation, and hypercoagulation, which are robust independent prognostic factors of progression to AIDS and comorbidities (23, 41, 42). To investigate the effect of the anticoagulant therapy on the outcome of a highly pathogenic SIVsab infection, we administered Ixolaris to five PTMs upon SIVsab infection (at the day of infection) and compared the natural history of SIVsab infection in Ixolaris-treated PTMs and untreated controls. Therefore, we first assessed the effects of the anticoagulant treatment by comparing the levels of immune activation and inflammation markers in PTMs receiving Ixolaris and in controls. The animals treated with Ixolaris showed significantly reduced levels of the proinflammatory cytokine IL-17 during early chronic infection (P = 0.03; Fig. 6C). The anticoagulant treatment also affected T cell immune activation, as demonstrated by a lower frequency of CD4+ T cells expressing HLA-DR+ and CD38+ (Fig. 6D), and by significantly lower frequency of CD8+ T cells expressing HLA-DR+ and CD38+ during early chronic infection (P < 0.001; Fig. 6E).
 
After treatment, TF expression on circulating CD14+ monocytes decreased compared with untreated controls (Fig. 6F). In addition, Ixolaris treatment significantly lowered both CD80 expression in chronically infected PTMs (P = 0.004; Fig. 6G) and CD86 expression in both acutely and chronically infected PTMs (P = 0.03; Fig. 6H) compared with the untreated controls. To further validate the reduction of monocyte activation induced by Ixolaris treatment, we measured glucose transporter-1 (Glut-1) expression on CD14+ monocytes, an important monocyte activation marker (43-45). Ixolaris-treated animals showed a significantly reduced Glut-1 expression after SIV infection compared to untreated controls (P = 0.001; Fig. 6I). Furthermore, we examined associations between expression of monocyte markers and lymphocyte activation using generalizing estimating equations, due to the nature of the data distribution and small sample size of the experimental groups. Using this approach, we found that the monocyte activation markers strongly associated with lymphocyte activation. CD80 and CD86 expression on CD14+ monocytes were directly associated with HLA-DR+ CD38+ expression on CD4+ T cells (P < 0.001 and P < 0.001, respectively), whereas Glut-1 expression on CD14+ monocytes was strongly associated with HLA-DR+ CD38+ expression on both CD4+ and CD8+ T cells (P < 0.001 and P < 0.001, respectively). Together, these results suggest that Ixolaris treatment had a beneficial effect in reducing immune activation and inflammation associated with SIV infection. The hypercoagulable status exhibited in SIVsab-infected progressive NHPs and HIV-infected patients is marked by elevated D-dimer, which is associated with increased incidence of cardiovascular comorbidities and mortality (21, 25, 46). Therefore, we next compared the D-dimer levels in Ixolaris-treated PTMs and controls. Ixolaris administration significantly reduced plasma D-dimer levels in acute SIVsab infection of PTMs (P = 0.033) (Fig. 6J) and resulted in lower D-dimer levels during early chronic infection, indicating a clear effect of Ixolaris in improving the coagulation status in the SIV-infected animals. To monitor the infection in SIVsab-infected PTMs, we measured plasma SIV viremia in all infected animals (47). The Ixolaris-treated group showed overall lower viral loads compared to controls (Fig. 6K).
 
Ixolaris-treated animals did not develop disease during the first 100 days after infection (Fig. 6L). In accordance with our previous studies in which two of five SIVsab-infected PTMs were rapid progressors (48, 49), one of three animals progressed to AIDS in the first 100 days after infection in the untreated group; however, no rapid progression was registered in the Ixolaris group. Statistical significance was not reached in the present study, possibly due to the small sample size. This result is of potential translational interest because it suggests that Ixolaris treatment may abrogate rapid progression in treated animals, likely due to the combined effect of reduced immune activation and inflammation, reduced hypercoagulable status, and small reduction in plasma viremia.
 
DISCUSSION
 
Chronic HIV infection has been associated with elevated circulating levels of biomarkers of coagulation, in particular D-dimer, and systemic inflammation, such as IL-6, sCD14, and CRP (22, 23, 50), which have been independently linked to a higher risk of non-AIDS-related death and mortality (23) even in persons treated with ART (51). Although ART-induced suppression of HIV viremia is shown to significantly reduce plasma levels of D-dimer, plasma levels remain higher than in non-HIV-infected populations (32). Monocytes have been previously described as immune cells involved in cardiovascular disease in both HIV− and HIV+ populations (4, 51-53) and are an important source of TF (9). Here, we performed a detailed immune profiling of monocyte subsets and delineated molecular signatures that characterize TF-expressing cells in the context of TLR activation. Our results demonstrated that TFpos monocytes exhibit elevated expression of several genes associated with innate immune activation. These findings argue that the subpopulation of monocytes that is able to up-regulate TF expression upon TLR4 activation is the classical subset defined as CD14+CD16−. Activated monocytes that do not express TF under the same circumstances exhibit markers of patrolling monocytes (CD14dimCD16+) such as up-regulation of CX3CR1 gene expression. In the setting of coagulopathy, it is possible that patrolling monocytes, expressing high levels of CX3CR1, a vascular homing receptor, might interact with thrombin clots. Our results indicate that the patrolling monocytes express the lowest level of PAR-1 and that PAR-1 expression is decreased by thrombin stimulation. Therefore, in vivo, it is possible that the interaction between CX3CR1-expressing patrolling monocytes and thrombin clots accounts for the lower expression of PAR-1. Furthermore, we demonstrated that the frequency of monocytes expressing TF upon activation could not be further increased with augmenting doses of TLR activation, suggesting an inherent capacity of these circulating monocytes to express this coagulation factor. We confirmed that nonmonocytic cells from peripheral blood are unable to express TF upon LPS stimulation. Flow cytometry-based phenotypic analysis revealed that compared to TFneg monocytes, TFpos cells displayed increased expression of HLA-DR and costimulatory molecules such as CD40 and CD86 as well as IL-6R. These results indicate a unique monocyte phenotype capable of producing TF. Previous flow cytometry-based studies have indicated that CD16+ monocytes are able to produce TF (54). However, our results indicate that the molecular signature associated with TF expression in monocytes is restricted to the classical proinflammatory subset that lacks expression of CX3CR1. It is possible that different experimental settings and/or gating strategies have resulted in discrepancies between the studies, and for this reason, we performed TF expression assessment in sorted monocyte subsets to identify the main TF producers. In addition, molecular gene analysis was performed in healthy individuals, and gene expression might differ in HIV+ individuals due to the trained immunity phenomenon (55). Our findings indicate that classical proinflammatory monocytes are the major subset capable of robustly expressing TF protein and also up-regulating TF functional activity upon LPS stimulation. Previous studies have indicated that TF expression on monocytes is increased in HIV-infected individuals compared to healthy controls and that the frequency of TFpos cells is associated with HIV disease progression (18). Here, we found that the frequency of TFpos monocytes, as well as TF functional activity, was similar between ART-naïve HIV+ individuals and those who achieved HIV suppression after ART initiation. These findings strongly indicate that TF expression by monocytes remains high in HIV+ individuals regardless of ART-induced virological suppression, which could be associated with increased potential to activate the coagulation cascade and cause cardiovascular disease. Consistent with this concept, previous work has demonstrated a positive correlation between frequency of TFpos monocytes and D-dimer levels in HIV+ patients (18). We validated these findings in our analysis of ART-naive HIV+ patients. Intriguingly, although the D-dimer plasma levels were significantly reduced after ART-induced suppression of HIV viremia, the frequency of TFpos monocytes remained elevated. It is plausible that some degree of coagulopathy persists despite the decreases in D-dimer and supports a more complex relationship between pro- and anticoagulant factors in untreated HIV due to poor synthetic liver function (20). This also suggests that assessing TF expression on monocytes would increase sensitivity for the detection of coagulopathy. Our findings further reveal TF as a potential therapeutic target in ART-treated patients with evidence of coagulopathy.
 
The pathogenic role of coagulation during progressive SIVsab infection in PTMs has been established previously (25), and NHPs serve as an important HIV model that can be controlled for key variables, such as time of infection. Here, we observed that compared to AGMs, which experience active viral replication but no disease progression, PTMs have significantly increased frequency of TF-expressing monocytes and TF functional activity upon chronic SIVsab infection. No differences were observed in TF expression and activity in vitro between PTMs and AGMs before SIV infection, suggesting that monocyte subsets from these two NHP species diverge markedly in response to chronic viral infection. These results also link the occurrence of SIV progression and systemic coagulopathy with increased capacity of monocytes to produce TF upon activation.
 
Aside from their role in coagulation, monocytes are also important in inflammatory processes due to their production of cytokines. There is a growing body of evidence indicating that persistent inflammation is associated with increased mortality in HIV (23, 56). Our experiments assessing polyfunctionality of monocytes by means of production of IL-1β, IL-6, and TNF-α upon TLR activation in vitro demonstrate that TFpos cells are more frequently triple cytokine producers when compared to TFneg monocytes in healthy individuals and in HIV+ patients, as well as in chronically SIV-infected PTMs. These results suggest that the same monocyte subset that expresses TF upon activation may also be implicated in persistent inflammation by producing multiple cytokines. This idea was reinforced by our findings demonstrating an increased frequency of polyfunctional monocytes in PTMs compared to AGMs after SIVsab infection but not in naïve animals. Thus, it is reasonable to propose that TFpos cells may be critically implicated in the promotion of systemic inflammation and coagulation associated with disease progression in chronic HIV and SIVsab infection (fig. S9).
 
Having identified a role of TF-expressing monocytes at the intersection of inflammation and coagulation, we hypothesized that interfering with TF could serve as a therapeutic approach to target hypercoagulation. Administration of Ixolaris in vivo decreased T cell activation as well as plasma IL-17 and D-dimer levels. Ixolaris produced no evidence of toxicity and was not associated with significant CD4+ or SIVsab viremia changes. The results of the in vivo study are significant for two reasons. First, they point to a causal relationship between coagulation and immune activation and inflammation. Thus, the Ixolaris treatment did not only reduce inflammation but also lowered monocyte and lymphocyte activation in the treated animals. The reduction of lymphocyte activation may be the direct result of reduced expression of the costimulatory markers CD80 and CD86 (two potent T cell activators) on monocytes. Alternatively, Ixolaris treatment could have affected the levels of immune activation by directly reducing T cell expression of PAR-1. Such a scenario is supported by our finding that Ixolaris treatment appears to have a stronger impact on the activation of CD8+T cell activation because these cells express higher levels of PAR-1. In vitro assays showed that Ixolaris treatment did not diminish TF expression on monocytes in response to LPS; however, these assays did not have the capacity to generate thrombin. The in vivo data show reduction of monocyte activation after Ixolaris treatment, highlighting the importance of TF-generated thrombin on monocyte activation.
 
Our study limitations included the small sample size of our longitudinal cohort and the cross-sectional nature of many experiments. In addition, the in vivo Ixolaris administration study included only a small number of ART-naïve acutely infected animals, prohibiting a more detailed evaluation of possible toxicity, drug interaction, complications, and potential survival benefit in untreated or treated animals. The impact of anticoagulant therapy on immune activation and inflammation in SIVsab-infected PTMs demonstrates that hypercoagulation is a significant source of persistent immune activation and inflammation in this model and probably in HIV-infected patients as well. Anticoagulant therapy by itself reduced important measurements of immune activation and inflammation and therefore has potential to improve the clinical management of HIV-infected patients. Second, we show that anticoagulant treatment improved the natural history of highly pathogenic SIVsab infection even in the absence of any other intervention aimed at either controlling viral replication (ART) or improving the health of the gut. Therefore, our study suggests that targeting the coagulation pathway in HIV-infected patients may be effective in reducing the immune activation and inflammation that are linked to cardiovascular comorbidities in HIV infection. These findings indicate that targeting TF may be used as a host-directed therapy in chronic HIV infection as well as other inflammatory diseases with similar immunopathology.

 
 
 
 
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