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HAART Controls Gut Microbes & decreases immune activation
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Microbial translocation is a cause of systemic immune activation in chronic HIV infection
Nature Medicine -Dec 2006 12, 1365 - 1371
Jason M Brenchley1, David A Price1, Timothy W Schacker2, Tedi E Asher1, Guido Silvestri3, Srinivas Rao4, Zachary Kazzaz1, Ethan Bornstein1, Olivier Lambotte5, Daniel Altmann6, Bruce R Blazar7, Benigno Rodriguez8, Leia Teixeira-Johnson8, Alan Landay9, Jeffrey N Martin10, Frederick M Hecht10, Louis J Picker11, Michael M Lederman8, Steven G Deeks10 & Daniel C Douek1
1 Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
2 Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA.
3 Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
4 Laboratory of Animal Medicine, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
5 University Hospital of Bicetre, Bicetre 94 276, France.
6 Department of Infectious Diseases, Hammersmith Hospital, Imperial College London, London W12 ONN, UK.
7 Department of Pediatrics, Division of Hematology, Oncology, and Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota 55455, USA.
8 Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44016, USA.
9 Department of Immunology and Microbiology, Rush Medical College, Chicago, Illinois 60612, USA.
10 University of California at San Francisco, San Francisco, California 90210, USA.
11 Vaccine and Gene Therapy Institute, Oregon Health and Science University, Portland, Oregon 97006, USA.
"....Immune activation decreases after initiation of highly active antiretroviral therapy (HAART) (see discussion in text of article below)....LPS levels decrease with HAART..."
Chronic activation of the immune system is a hallmark of progressive HIV infection and better predicts disease outcome than plasma viral load, yet its etiology remains obscure. Here we show that circulating microbial products, probably derived from the gastrointestinal tract, are a cause of HIV-related systemic immune activation. Circulating lipopolysaccharide, which we used as an indicator of microbial translocation, was significantly increased in chronically HIV-infected individuals and in simian immunodeficiency virus (SIV)-infected rhesus macaques (P 0.002). We show that increased lipopolysaccharide is bioactive in vivo and correlates with measures of innate and adaptive immune activation. Effective antiretroviral therapy seemed to reduce microbial translocation partially. Furthermore, in nonpathogenic SIV infection of sooty mangabeys, microbial translocation did not seem to occur. These data establish a mechanism for chronic immune activation in the context of a compromised gastrointestinal mucosal surface and provide new directions for therapeutic interventions that modify the consequences of acute HIV infection.
Chronic immune activation is a characteristic feature of progressive HIV disease. Indeed, polyclonal B-cell activation was one of the first described immunological abnormalities in HIV-infected individuals1. Subsequently, increased T-cell turnover2, increased frequencies of T cells with an activated phenotype3, and increased serum levels of proinflammatory cytokines and chemokines4 were observed. Notably, the degree of immune activation is a better predictor of disease progression than plasma viral load5. However, the underlying causes of immune activation have remained elusive6, 7.
Recently, there has been a substantial reappraisal of the tempo and anatomical location of pathogenic events in the course of HIV disease: the bulk of CD4 T-cell depletion occurs rapidly within the first few weeks of infection and is predominantly localized to the gastrointestinal tract8, 9, 10, 11, 12. Thus, there is an early breach in the integrity of the mucosal immune system. Notably, the extent of mucosal CD4 T-cell depletion in pathogenic SIV infection of rhesus macaques determines the rate of progression to AIDS (ref. 13). This has led to the suggestion that injury to the immune component of the gastrointestinal mucosal surface, along with damage to the intestinal epithelial microenvironment14, 15, 16 with its antimicrobial functions17, may effect systemic immune activation during the chronic phase of HIV infection through the increased translocation of luminal microbial products6. Indeed, the phenomenon of microbial translocation, defined as the translocation of microbes and/or microbial products without overt bacteremia, occurs after damage to the gastrointestinal tract during conditioning for hematopoietic transplantation, resulting in systemic immune activation and exacerbation of graft-versus-host disease (GVHD)18. The gastrointestinal tract is the principal source of such microbial products because of its massive bacterial load compared to other anatomical sites17, 18. Moreover, microbial translocation is a noted feature of inflammatory bowel disease (IBD)19. Lipopolysaccharide (LPS), a major component of Gram-negative bacterial cell walls and a potent immunostimulatory product20, can be quantitatively assessed in plasma and LPS levels are commonly measured to determine the degree of microbial translocation in GVHD and IBD (refs. 18,19,21, 22, 23, 24). In addition, plasma LPS levels are directly associated with the degree of intestinal permeability following invasive gastrointestinal surgery25. In the study described here, we investigated whether microbial translocation is a feature of chronic HIV infection that contributes to systemic immune activation.
Results
Raised plasma LPS in HIV infection
We initially quantified plasma levels of LPS in HIV-infected and uninfected humans and in SIV-infected and uninfected nonhuman primates (286 subjects). Chronically HIV-infected individuals and individuals with AIDS (<200 CD4 T cells per l) had significantly higher plasma LPS levels than uninfected individuals (P < 0.0001, Fig. 1a). The chronically HIV-infected cohort and AIDS cohort did not have considerably different LPS levels compared to each other and together are classified as progressors henceforth. This observation is strongly suggestive of increased microbial translocation. The sources of plasma LPS could include, but are not limited to, commensal and pathogenic bacteria and even subclinical opportunistic infections. The latter are difficult to define, however, as they have no clinical manifestations. Notably, no individual had any overt signs of bacteremia and those with AIDS had no evidence of opportunistic infections. Even though mucosal CD4 T-cell depletion occurs during the acute phase of the infection8, 9, 11, plasma LPS levels had not yet increased in the acute/early cohort (within 4 weeks of seroconversion), suggesting that the consequences of depletion and mucosal damage are not manifest until the chronic phase or that there is transient mobilization of factors that neutralize circulating LPS, as discussed below.
To show that increased plasma LPS in HIV-infected individuals could be attributed to translocation from the gastrointestinal tract, we aimed to decrease intestinal bacterial load with an extended 'bowel-sterilizing' antibiotic regimen in SIV-infected rhesus macaques, which usually progress rapidly to AIDS and manifest high levels of immune activation. First we confirmed that macaques have increased plasma LPS levels after SIV infection by sampling 11 macaques before and 100 d after infection. Plasma LPS increased after infection in all but one macaque (Fig. 1b). To test the hypothesis that the source of the translocated LPS was the massive bacterial load within the gastrointestinal tract, we then treated two SIV-infected macaques, which showed no signs of sepsis or bacterial infection, with antibiotics for 2 weeks and monitored fecal bacterial loads and plasma LPS levels. After 1 week of antibiotic treatment, plasma LPS levels decreased markedly in both macaques (Fig. 1c), concomitant with a substantial decrease in major fecal Gram-negative bacteria (Table 1). These data suggest that the origin of plasma LPS in SIV-infected rhesus macaques and, by inference, in HIV-infected humans is translocation from the gastrointestinal tract. Moreover, these data are consistent with previous reports suggesting that the source of plasma endotoxemia in IBD is microbial translocation from the gastrointestinal tract23, 24. In addition, it has recently been shown that by 28 d after SIV infection of rhesus macaques, multiple microabscesses are histologically apparent in the small bowel, indicating local bacterial invasion of the mucosal surface (A.T. Haase, personal communication). Unfortunately, by week 2 of our regimen, plasma LPS levels had increased as gastrointestinal outgrowth of other bacterial species became apparent (Table 1). This transient and incomplete clearance of gastrointestinal bacterial load would explain why we observed only a modest decrease in the measures of LPS-mediated in vivo monocyte stimulation discussed below (data not shown).
Chronic LPS stimulation in vivo
CD14+ monocyte/macrophages secrete soluble CD14 (sCD14), which binds LPS (ref. 26), and proinflammatory cytokines such as tumour-necrosis factor (TNF) and interleukin-1 (IL-1) upon LPS stimulation. Thus, to establish evidence for direct chronic LPS stimulation in vivo, we measured plasma sCD14 levels. We also measured plasma LPS-binding protein (LBP), which is produced by gastrointestinal and hepatic epithelial cells in response to LPS stimulation. We found significantly higher levels of plasma sCD14 in the acute/early cohort and in the progressors, as compared to uninfected individuals (P < 0.0001, Fig. 2a), consistent with a previous report27. In addition, in the progressors, we found a significant correlation between plasma LPS and sCD14 (r = 0.3, P = 0.001), suggesting that LPS directly stimulates sCD14 production in vivo. Previous reports suggest that the HIV glycoprotein gp120 could stimulate monocytes to produce sCD14 in vitro27; however, we found no correlation between sCD14 and plasma viral load (r = 0.05, P = 0.5). Plasma LBP levels were also significantly increased in HIV-infected individuals (P = 0.0099, Fig. 2b) and correlated positively with sCD14 levels (Supplementary Fig. 1 online). If CD14+ monocyte/macrophages are stimulated chronically in vivo, they become refractory to further stimulation with LPS in vitro28. Indeed, we found a significant inverse correlation between the ability of blood monocytes to respond to in vitro LPS stimulation and plasma levels of LPS (P = 0.017, r = -0.53, Fig. 2c). These data indicate that the increased circulating LPS is bioactive in vivo and may also contribute to functional impairment of monocytes. However, monocytes from HIV-infected and uninfected individuals were similarly responsive to stimulation with TNF and interferon- (IFN-), suggesting that monocyte hyporesponsiveness to LPS is a specific consequence of Toll-like receptor (TLR)-mediated chronic stimulation (data not shown).
Naturally occurring immunoglobulin-M (IgM), immunoglobulin-A (IgA) and immunoglobulin-G (IgG) antibodies to the LPS core oligosaccharide neutralize LPS activity, are probably produced by T-dependent B cells and can be measured in healthy human plasma29, 30. In conditions of acute microbial translocation such as sepsis, these antibodies, termed endotoxin-core antibodies (EndoCAb), bind to and clear LPS from the circulation, and their titers decrease29. We found significantly lower EndoCAb titers in the acute/early cohort compared to uninfected individuals (P < 0.0001, Fig. 2d). This observation suggests that in the context of normal LPS levels but raised sCD14 levels (Figs. 1a and 2a), microbial translocation occurs during the early phase of infection but that translocated LPS is bound and neutralized by circulating EndoCAb. In conditions of chronic microbial translocation such as IBD, EndoCAb levels are increased31 presumably as part of a healthy humoral response to LPS. Notably, we found that EndoCAb levels were significantly lower in progressors compared to either uninfected individuals (P < 0.0001) or the acute/early cohort (P = 0.0002) (Fig. 2d). This suggests that in chronic HIV infection, EndoCAb levels are insufficient to neutralize circulating LPS and prevent systemic immune activation, a defect that may be attributable to the dysfunctional B-cell responses in chronic HIV infection32. Indeed, in our HIV-infected cohorts, we found a significant inverse correlation between plasma EndoCAb titers and LPS levels (P = 0.0005, r = -0.319, Fig. 2e), consistent with this explanation.
LPS levels and chronic immune activation
Although LPS is a potent immunostimulatory product per se, we measured LPS as an indicator of microbial translocation. Multiple products such as RNA, DNA, peptidoglycan and flagellin20 derived from bacteria, viruses, fungi and other gastrointestinal residents might translocate and cause systemic immune activation. We examined whether LPS levels were associated with manifestations of immune stimulation that are not directly related to LPS-mediated activation. We found a significant positive correlation between plasma IFN- and plasma LPS levels (P < 0.0001, r = 0.624, Fig. 3a). As plasmacytoid dendritic cells (pDCs), the predominant source of IFN- in vivo33, do not express TLR4 or respond to LPS, it is likely that increased LPS levels are indicative of an increase in other circulating factors derived from gastrointestinal flora that can directly stimulate pDCs (ref. 33). We could not detect raised plasma levels of IL-1 or TNF (data not shown), which, in previous studies, have been found to be increased only in advanced AIDS or opportunistic infections34, 35, 36. Clearly, HIV itself is a candidate for an innate immunostimulatory agent in infected individuals, as its RNA genome can stimulate pDCs in vitro through TLR7 (ref. 37). However, we found no correlation between plasma viral load and IFN- (r = 0.2, P = 0.2). Additionally, we found a significant positive correlation between plasma LPS levels and the frequency of circulating CD8 T cells with an activated CD38+ HLA-DR+ phenotype (P < 0.03, Fig. 3b,c) suggesting that the products of microbial translocation might directly38, or indirectly through the effects of cytokines and chemokines, result in polyclonal T-cell activation. Consistent with this hypothesis, we could induce low levels of T-cell, B-cell and monocyte activation by culturing lymphocytes from HIV-uninfected individuals with plasma from HIV-infected individuals with high LPS levels (Supplementary Fig. 2 online).
LPS levels decrease with HAART
Immune activation decreases after initiation of highly active antiretroviral therapy (HAART), although at a much slower rate than the decrease in viral load39, 40. Reduction in viral replication might allow reconstitution of the immunological and structural barriers that prevent microbial translocation, and, consequently, plasma LPS levels would decrease with HAART. We therefore measured plasma LPS in 28 HIV-infected individuals before and 48 weeks after initiation of HAART when plasma viral loads were undetectable. We found that plasma LPS levels decreased in all but four individuals (Fig. 3d). Furthermore, there was a significant inverse correlation between reconstitution of blood CD4 T cells and the plasma LPS level in individuals undergoing HAART (P = 0.0151, r = -0.463, Fig. 3e). These data point to a central role for HIV itself in perpetuating microbial translocation, such that ongoing infection and depletion of CD4 T cells throughout the chronic phase of the disease prevents the re-establishment of competent immunological control of microbial translocation. The complement to such events would be that microbial translocation in its turn perpetuates viral replication through the provision and activation of target T cells for HIV (ref. 41). However, the relationships between HIV replication, microbial translocation and LPS levels, and CD4 T-cell reconstitution are likely to be complex and nonlinear, as we found no correlation between LPS levels and either absolute CD4 count (P = 0.226) or plasma viral load (P = 0.492). Indeed, plasma LPS levels in the HAART cohort remained significantly above those in the uninfected cohorts (P = 0.0026, Fig. 3d), suggesting that microbial translocation may still occur, albeit to a lesser degree, owing to persistent abnormalities at the gastrointestinal mucosal surface and only limited reconstitution of gastrointestinal CD4 T cells on therapy10, 12, 14. In support of this notion, we also found that sCD14 levels were unchanged after 48 weeks of HAART compared to those at treatment-nave time points (P = 0.87), even though plasma viral loads were undetectable. Moreover, EndoCAb titers remained low on HAART (data not shown), possibly due to persistent B-cell abnormalities that are only slowly reversed with HAART (ref. 42) and that may consequently aggravate the prolonged plasma endotoxemia.
HIV-infected controllers
Few HIV-infected individuals, often termed 'elite controllers', maintain low or undetectable plasma viral loads without treatment and have low levels of immune activation43. Immune-mediated suppression of HIV replication may account for nonprogression in some individuals who express HLA-B57, HLA-B58 or HLA-B27. However, among the mechanisms that might also prevent disease progression in this probably heterogeneous group, reduced microbial translocation, more effective control of microbial products and/or an attenuated immune response to microbial products44 could diminish immune activation and viral replication, and slow disease progression. We found that HIV-infected controllers had significantly higher plasma LPS levels than uninfected individuals (P < 0.0001), but lower levels than progressors that trended to but did not achieve significance (P = 0.0647) (Fig. 4a). This suggests that microbial translocation is increased in controllers with undetectable viremia, consistent with the finding that such individuals have mucosal CD4 T-cell depletion and intestinal epithelial damage45. However, we also found that plasma levels of sCD14 (Fig. 4b), which correlated with plasma LPS in this cohort (r = 0.5 P = 0.04), and LBP (Fig. 4c) were significantly lower (P < 0.002) and plasma EndoCAb titers (Fig. 4d) significantly higher (P = 0.016) in controllers than among progressors. Furthermore, EndoCAb levels did not seem to be reduced at higher levels of LPS, as was the case with progressors (Supplementary Fig. 3 online). Taken together, these data suggest that in certain controllers the immunostimulatory effects of LPS are attenuated, and more effective neutralization of plasma LPS by consistently maintained higher titers of circulating EndoCAb may serve to reduce immune activation and slow disease progression. Indeed, increased EndoCAb titers predict better clinical outcome in individuals with sepsis syndrome29.
Nonpathogenic natural SIV infection
Chronic SIV infection in its natural primate hosts, such as sooty mangabeys, is typically nonpathogenic, even in the context of chronic high-level viremia, and is characterized by low levels of immune activation46. To establish a possible mechanism for this outcome, we investigated whether microbial translocation occurred during chronic SIV infection of sooty mangabeys. In marked contrast to chronically HIV-infected humans and SIV-infected rhesus macaques, we found that SIV-infected and uninfected sooty mangabeys had similarly low levels of plasma LPS (Fig. 4e), Additionally, we found no differences in plasma sCD14 levels (Fig. 4f) or EndoCAb titers (Fig. 4g) between these cohorts. These findings suggest that microbial translocation does not occur in this nonpathogenic infection. As chronically SIV-infected sooty mangabeys consistently show low immune activation, these data support a direct link between microbial translocation and the establishment of a state of generalized immune activation during a primate lentiviral infection. The mechanisms by which the mucosal barrier is preserved in nonpathogenic SIV infection of sooty mangabeys are unclear, as depletion of mucosal CD4 T cells, although not as pronounced as in rhesus macaques, has been observed during acute infection (G.S., unpublished observations). It is likely, therefore, that sooty mangabeys and perhaps other natural hosts have evolved to preserve the integrity of their mucosal barrier and prevent microbial translocation in a way that is less dependent on CD4 T cells.
Discussion
To summarize, in HIV-infected humans with progressive disease, we found (i) significantly raised levels of plasma LPS as an indicator of increased microbial translocation (P < 0.0001); (ii) evidence for chronic in vivo stimulation of monocytes by LPS; (iii) an association between raised plasma LPS and measures of innate and adaptive immune activation; (iv) a decrease in plasma LPS upon treatment with HAART; and (v) an association between reduction in plasma LPS and CD4 T-cell reconstitution with HAART. In rhesus macaques, we found an increase in plasma LPS levels after infection with a pathogenic SIV strain, and a transient but marked decrease in plasma LPS on treatment with a bowel-sterilizing antibiotic regimen. Taken together, these findings suggest that the increased translocation of gastrointestinal microbial products directly contributes to systemic immune activation in the chronic phase of HIV infection and may ultimately determine the rate of progression to AIDS. This model is consistent with the relatively slow progression of the chronic phase of HIV infection, which is determined by the degree of immune activation, in marked contrast to the precipitously rapid destructive events at the mucosal surfaces during the acute phase7. It seems likely that the pathogenic events, which include, but might not be restricted to, massive early mucosal CD4 T-cell depletion9, 10, 11, 12 and enteropathy15, 16, are responsible for compromising the integrity of the mucosal barrier. The reduction in plasma LPS levels with HAART suggests that HIV replication plays a central role in perpetuating microbial translocation and its consequences probably by preventing immune reconstitution at mucosal surfaces and reducing immune control of circulating microbial products.
The causes of immune activation observed during the acute, in contrast to the chronic, phase of infection may include factors in addition to translocated microbial products, which might explain the dissociation between plasma LPS and immune activation in acute/early infected individuals. A prime candidate would be HIV itself, the plasma levels of which are orders of magnitude higher in the acute phase than in the chronic phase. Hence, as in other acute viral infections, such as Epstein-Barr virus47 or influenza48, the immune activation that occurs during acute HIV infection may be also attributed, at least in part, to the direct consequences of high viral loads per se. In addition, the proinflammatory environment that occurs locally in the gastrointestinal tract in HIV infection45 may in turn cause further damage to the epithelial barrier, thus augmenting microbial translocation and subsequently fueling systemic immune activation.
In slow or nonprogressive infection, different mechanisms may operate to mitigate the translation of these destructive mucosal events into disease. Indeed our results suggest that in controllers, raised plasma LPS levels may be offset by persistently higher titers of EndoCAb. However, in nonpathogenic SIV infection of sooty mangabeys, the mucosal barrier seems to remain sufficiently intact so as to prevent microbial translocation. Aside from immunological control of HIV/SIV replication, other host genetic factors may result in a nonprogressive disease course. These factors could include many of the genes involved in the host inflammatory response, such as the genes encoding TLRs, functional polymorphisms of which may bestow reduced responsiveness to the microbial stimuli that trigger immune activation44. It is in this context that our data provide new directions for therapeutic interventions. The aim would be to prevent or reduce the propagation of HIV at mucosal surfaces14, 49, to restore the immunological and epithelial integrity of the mucosal barrier14 and to block the cellular and molecular pathways through which microbial products cause systemic immune activation50.
Methods
Subjects.
We recruited 205 HIV-infected and 47 HIV-uninfected individuals. We grouped HIV-infected individuals into disease categories as follows: acute/early, based upon known seroconversion within 4 weeks of sampling (n = 50); elite controllers, based on undetectable plasma viremia without HAART and CD4 counts greater than 500 cells/ l (n = 30); chronic, based upon plasma viremia above 400 copies/ml and CD4 counts greater than 200 cells/l (n = 59); and AIDS, based upon CD4 counts less than 200 cells/l (n = 37). We grouped the chronic and AIDS subjects together and considered them as progressors for some analyses. We obtained subjects from several unique cohorts, and denoted them in our figures by colored symbols that indicate both HIV status and cohort. We recruited 15 HIV unexposed individuals (black circles) from the US National Institutes of Health (NIH). We recruited 32 exposed uninfected individuals (open circles), 50 acutely infected individuals (orange circles), 27 chronically infected individuals (red circles), 31 individuals with AIDS (pink circles), and 17 elite controllers (yellow circles) at the University of California at San Francisco. We recruited 31 individuals with chronic infection (green circles) and 7 individuals with AIDS (turquoise circles) participating from multiple cites in the AIDS Clinical Trials Group protocol 5014 before and 48 weeks after initiation of HAART. We recruited 19 individuals with chronic infection (blue circles) at the University of Minnesota. We recruited 10 individuals with chronic infection (brown circles) and 13 elite controllers (purple circles) at the department of Infectious Diseases of the University Hospital of Bicetre and from the Agence Nationale de Recherches sur le SIDA (ANRS), French Seroconversion Cohort (SEROCO), French Hemophiliac Cohort (HEMOCO). We collected plasma samples after centrifugation of blood drawn into either EDTA- or acid citrate dextrose (ACD)-treated collection tubes, and obtained peripheral blood mononuclear cells (PBMC) by density centrifugation. We determined plasma viral loads using standard assays at each center. No HIV-infected individual had overt clinical manifestations of or were receiving treatment for bacterial infections.
We infected six colony-bred healthy rhesus macaques (black squares) housed at Bioqual Inc. with 100 animal infectious doses of uncloned pathogenic SIVMAC251 intravenously. We infected 5 colony-bred healthy rhesus macaques housed at the Oregon National Primate Research Center with 5-ng equivalents of SIV p27 intravenously, 3 with SIVMAC239 and 2 with SIVMAC155T3. Macaques were housed in accordance with American Association for Accreditation of Laboratory Animal Care guidelines and were sero-negative for SIV, simian retrovirus and simian T-lymphotrophic virus (STLV)-1 before the study. We collected plasma and samples before infection and around 100 d after infection. We included 12 naturally SIV-infected and 11 uninfected sooty mangabeys (black triangles) in this cross-sectional analysis. In uninfected sooty mangabeys, negative SIV PCR of plasma and negative HIV-2 serology confirmed the absence of SIV infection. All sooty mangabeys were housed at the Yerkes National Primate Research Center and maintained in accordance with NIH guidelines.
All human subjects gave informed consent and all studies were approved by the appropriate Institutional Review Boards or Animal Care and Use Committees.
We performed the described assays on as many samples as possible with the investigator blinded as to the identity of the samples. However, as samples were often limiting, not all assays could be performed on all samples.
LPS levels.
We diluted plasma samples to 20% with endotoxin-free water and then heated them to 70 C for 10 min to inactivate plasma proteins. We then quantified plasma LPS with a commercially available Limulus Amebocyte assay (Cambrex) according to the manufacturer's protocol. We ran samples in duplicate and subtracted the background.
Immune activation.
We used commercially available ELISAs to quantify plasma levels of sCD14, IFN- (R&D Systems), LBP and EndoCAb (Cell Sciences). We performed each ELISA in duplicate and according to the manufacturers' protocols. We determined the activation of CD8 T cells by measuring the expression of CD38 and HLA-DR by gated CD3+CD8+ cells, using flow cytometry after staining PBMC with fluorochrome-conjugated antibodies to CD3, CD8, CD38 and HLA-DR (Becton Dickinson). We determined the ex vivo responses of blood monocytes to LPS by stimulating PBMC with 1 g/ml purified LPS (Sigma) for 5 h in the presence of 10 g/ml Brefeldin A (Sigma). We then stained cells with fluorochrome-conjugated antibodies to CD14 and HLA-DR, and then permeablized and stained intracellularly with fluorochrome-conjugated antibodies to TNF and IL-1 (Becton Dickinson). After analysis by flow cytometry, we gated monocytes based upon the coexpression of HLA-DR and CD14, and we determined the percentage of monocytes producing TNF and IL-1. We performed flow cytometry with an LSRII or FACS Calibur (BD), and performed instrument set-up according to the manufacturer's recommendations.
Antibiotic treatment.
We treated two therapy-naive, chronically SIV-infected rhesus macaques for 2 weeks with a combination of neomycin (10 mg per kg body weight orally), metronidazole (40 mg/kg orally) and cefotaxime (150 mg/kg intramuscularly) daily.
Statistical analysis.
We performed Spearman's rank-correlation, Wilcoxon matched-pairs and Mann-Whitney U-tests using Prism 4.0 software (Prism).
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