During established HIV-1 infection, an increase in plasma bacterial lipopolysaccharide (LPS) levels has been proposed to reflect microbial translocation from the gut serving as a potential inflammatory stimulus accelerating disease pathogenesis . The level and effect of circulating LPS can be modulated by LPS-binding protein (LBP) , soluble CD14 (sCD14) [3–5], naturally occurring antibodies to the LPS core oligosaccharide [endotoxin-core antibodies (EndoCAb)] [6,7] and plasma gelsolin (which can bind actin and LPS) [8–12]. The interplay between antiretroviral therapy (ART) interruption-mediated viremic episodes and changes in plasma levels of LPS and its host-derived ligands remains unknown and is the subject of this study.
Patients and methods
Cryopreserved plasma from 26 uninfected and 51 HIV-positive patients was analyzed. Ten out of the 51 HIV-positive participants were viremic ART-naive [median, 25th–75th interquartile range (IQR) plasma HIV-1 RNA (viral load)=19601 copies/ml (11545, 69954); median (IQR) CD4 cell count = 273.5 cells/μl (191.25, 347)] and 41 of 51 HIV-positive participants were chronically suppressed patients participating in a parent study [recruitment characteristics: age ≥18 years, presence of ART (three or more drugs), CD4 cell count >400 cells/μl with a history of nadir CD4 cell count ≥100 cells/μl, and viral load <50 copies/ml with a >6 months history of <500 copies/ml] and followed under continuous therapy (n = 20) or repeated therapy interruptions (n = 21) before undergoing an open-ended therapy interruption. Patient demographics for the parent study are described in our prior publications [13,14].
Samples from uninfected and viremic ART-naive HIV-positive patients were analyzed at a single visit, whereas available samples from the parent study were analyzed as follows: at baseline (viral load <50 copies/ml on ART, n = 41), on continuous ART for a 40-week follow-up (n = 16), during a 6-week therapy interruption (n = 21), and during open-ended therapy interruption: after patients reached viral set point (average viral load of the first three consecutive measurements of viral load with <0.5 log variation, n = 21) and at the last available viremic time point of the open-ended therapy interruption (n = 9).
Informed consent was obtained according to the Human Experimentation Guidelines of the US Department of Health and Human Services and of the authors' institutions. The study protocol was approved by the Institutional Review Boards of the Wistar Institute and Philadelphia FIGHT.
Lipopolysaccharide levels and immune activation
LPS levels were determined in duplicate by the limulus amebocyte assay according to the manufacturer's protocol (Cambrex Bioscience, Walkersville, Maryland, USA) in plasma samples diluted 1/100 (dilution determined by product inhibition test) with endotoxin-free water and heated to 70°C for 10 min to inactivate plasma proteins. Plasma levels of sCD14 (R&D, Minneapolis, Minnesota, USA), LBP (Cell Sciences, Canton, Massachusetts, USA), IgM EndoCAb (Cell Sciences), and IFN-α (PBL Biomedical Laboratories, Piscataway, New Jersey, USA) were determined by enzyme-linked immunosorbent assay (ELISA) as per manufacturer's specifications. Measurements were run in duplicate on a kinetic absorbance reader at 450 nm (Rainbow Reader; SLT-Lab Instruments, Grodig/Salzburg, Austria). Lower limits for LPS, sCD14, LBP, EndoCAb and IFN-α were 0.1 EU/ml, 250 pg/ml, 781.25 pg/ml, 0.054 MMU/ml and 12.5 pg/ml, respectively. Plasma levels of gelsolin were determined by immunoblotting using monoclonal antihuman gelsolin antibody (G4896; Sigma, St Louis, Missouri, USA) as described . Whole blood flow cytometry was used for assessment of T-cell activation (CD8+/CD38+, CD8+/HLA-DR+ and CD3+/CD95+) as described .
Data are presented as medians with 25th–75th IQR in parenthesis. Variable distributions were analyzed for normality using the Shapiro–Wilk W test (P > 0.05). Depending on data distribution, between groups comparisons were performed by t-test or the Wilcoxon/Kruskal–Wallis test (rank sums), whereas between time points comparisons were performed using nonparametric Wilcoxon sign-rank test or paired t-tests. Associations between variables were assessed using Spearman or pairwise correlation tests. All statistical tests were performed using JMP4 (SAS Institute, Cary, North Carolina, USA).
Increased plasma lipopolysaccharide levels observed only after long-term HIV-1 replication
Initial cross-sectional analysis showed that ART suppressed HIV-1-positive patients had lower LPS levels compared with uninfected participants (P < 0.0001), whereas ART-naive viremic patients had higher LPS levels compared with ART-suppressed HIV-1-positive patients (P = 0.0003) (Fig. 1a).
Longitudinal analysis showed no change in LPS levels during continuous ART, as well as during therapy interruptions of less than 12 weeks, including analysis during a 6-week therapy interruption [median (IQR) viral load = 10 745 copies/ml (2527, 61 874), Fig. 1b] and during open-ended therapy interruption when viral set point was reached [median (IQR) duration = 9 weeks (8, 12); median (IQR) viral load = 11 067 copies/ml (2851, 26 259)]. In contrast, steady-state viremia for more than 12 weeks resulted in significantly increased LPS levels (P = 0.0171, Fig. 1c), as shown by higher LPS levels at the last available viremic time point during open-ended therapy interruption [median (IQR) duration = 19 weeks (12, 35); median (IQR) viral load = 43 748 copies/ml (23 192, 101 044)] compared with levels at start of the open-ended therapy interruption (viral load <50 copies/ml). This differential effect of less than 12 versus more than 12 weeks of viral replication on LPS levels was observed despite a lack of significant difference in viral load. The impact of prolonged viremia was also supported by the longitudinal change of LPS in patients followed over sequential 6 week to more than 12 weeks therapy interruptions (P = 0.0009, n = 7, Fig. 1d) and by cross-sectional analysis showing higher LPS levels in patients with steady-state viremia for more than 12 weeks therapy interruption compared with uninfected participants (P < 0.0001) or ART-suppressed HIV-1-positive patients (P = 0.031).
Lack of association between onset of immune activation and plasma lipopolysaccharide levels
In agreement with previous studies, we observed a significant rise in the frequency of CD3+/CD95+ (P = 0.0022, n = 21), CD8+/CD38+ (P < 0.0001, n = 20, Fig. 1e) and CD8+/HLA-DR+ (P = 0.001, n = 20) T cells concurrent with the onset of viral replication during the 6-week therapy interruption. Change in the frequency of activated T cells after therapy interruption was observed despite lack of changes in LPS levels (Fig. 1b). No correlation between LPS levels and T-cell activation or plasma levels of IFN-α following therapy interruption was observed at any time point analyzed.
Binding of lipopolysaccharide by endotoxin-core antibodies and sCD14 during therapy interruption-mediated viral rebound
To investigate potential reasons for a lack of change in LPS levels after short-term viremia, plasma levels of LPS-binding molecules (EndoCAb, sCD14, LBP, gelsolin) were measured on the same samples.
A constitutive role for EndoCAb in modulating LPS levels was supported by a negative correlation between LPS and EndoCAb levels in uninfected participants (correlation = −0.430, P = 0.0282). Activation of an effective LPS clearance response by circulating EndoCAb during less than 12 weeks therapy interruption was suggested by a negative association between viral load at week 6 of therapy interruption (correlation = −0.502, P = 0.0204) and the change of EndoCAb (ΔEndoCAb) from start to end of the 6-week therapy interruption (Fig. 2a). A negative association between viral load and LPS levels at week 6 of therapy interruption (Spearman's Rho = −0.612, P = 0.0152) was also observed (Fig. 2a) concurrent with a lack of rise in LPS (Fig. 1). A strong negative association between ΔEndoCAb and ΔLPS (correlation = −0.851, P = 0.0073) was present from time on therapy to a subsequent steady-state viremic time point during less than 12 weeks therapy interruption (Fig. 2b), whereas no correlation between EndoCAb and LPS was observed after more than 12 weeks therapy interruption.
In contrast to EndoCAb, sCD14 showed a positive association with LPS (Fig. 1f). In addition, sCD14 showed changes similar to those observed for LPS, such as higher levels in steady-state viremic patients for more than 12 weeks therapy interruption (P = 0.02) or ART-naive patients (P = 0.0006) compared with uninfected participants and in ART-naive patients (P < 0.0001) compared with ART-suppressed HIV-1-positive patients (data not shown). Levels of sCD14 did not change during continuous ART or a 6-week therapy interruption, but increased during long-term therapy interruption for more than 12 weeks compared with levels during ART (P < 0.0001), further suggesting sCD14 to be a correlate to LPS levels. A negative correlation between ΔsCD14 and ΔEndoCAb (correlation = −0.896, P = 0.0025) during a 6-week therapy interruption was also observed.
Unexpectedly, no difference among groups or change during viremia was demonstrated for LBP or gelsolin, beside lower levels of LBP in steady-state viremic patients after more than 12 weeks therapy interruption compared with uninfected participants (P = 0.01).
We found different effects of viral replication on LPS plasma levels possibly dependent on the duration of the viremic episode and the degree of plasma LPS clearance by its ligands over time. No association between plasma LPS levels and the onset of immune activation during therapy interruption-associated viral replication was observed.
Increased EndoCAb IgM levels are common in conditions of chronic microbial translocation in the presence of functional B-cell responses , whereas acute microbial translocation coincides with lower levels of EndoCAb [7,17,18]. Decreases in EndoCAb levels observed in our study during viral replication of less than 12 weeks suggest that clearance of LPS from the circulation during short-term viremia is mediated by EndoCAb. A rise in LPS levels during long-term viremia of more than 12 weeks is likely attributed to EndoCAb saturation by excess of LPS or to inadequate B-cell function .
Consistent with previous studies showing increased levels of sCD14 in trauma , sepsis [21,22], autoimmune diseases [23,24], and HIV-1 infection [1,25,26], we observed a rise of sCD14 plasma levels in the presence of viral replication. In contrast to EndoCAb, no negative association with LPS was found, suggesting an inactivating role for sCD14 in the presence of LPS. In contrast to EndoCAb or sCD14, no changes in plasma levels of LBP and gelsolin were observed at any time point. These findings could be explained by differences in affinity, production, or clearance of these other LPS-binding molecules, or perhaps by the low molar ratio of LPS to these relatively abundant ligands.
Brenchley et al.  reported decreased LPS levels in the presence of ART and a positive association between immune activation and plasma LPS levels in chronically HIV-infected patients and in patients with AIDS. We not only confirmed their finding in patients with HIV viremia, but also observed lower LPS levels in suppressed patients compared with uninfected participants. This last counterintuitive finding may potentially reflect independent effect of ART on LPS levels or gut flora. Use of antibiotic prophylaxis by our suppressed patients could have contributed to this finding, and this type of information should be collected in targeted future studies to address this possibility. In addition to effective inactivation of LPS effects by EndoCAb in the short term, the observed lack of association between LPS levels and initial changes in immune activation following therapy interruption may be attributable to characteristics of our cohort such as lack of progression to AIDS, prior exposure to ART resulting in control of immune activation during suppression, or limited exposure to viral replication. Importantly, our data do not exclude the fact that T-cell activation during therapy interruption-mediated viremia may be affected by microbial translocation in addition to direct HIV-1 effects, as this study did not address measurement of other microbial products or their direct effects on T cells [27,28] or both.
Taken together, our data indicate that control of plasma microbial product translocation is only transiently maintained in the face of de-novo viral replication following therapy interruption and is dependent on the duration of the viremic episode and EndoCAb levels. This finding may help explain the lack of a positive association between LPS levels and viral load observed by others and us. The mechanism and capacity of LPS clearance achievable during therapy interruption following immune reconstitution remain to be further elucidated.
We would like to thank the uninfected participants and the HIV-1-positive patients who participated in the study and their providers, Cecile Gallo and Agnieszka Mackiewicz for study assistance, Jane Shull and the Board and Staff of Philadelphia FIGHT for providing patients samples, and Anne Meibohm for manuscript review. This work was primarily supported by a grant to L.J.M. by the National Institute of Allergy and Infectious Disease NIH AI48398. Additional support was provided by The Philadelphia Foundation (Robert I. Jacobs Fund), The Stengel–Miller family, AIDS funds from the Commonwealth of Pennsylvania, and from the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health, as well as by the Cancer Center Grant (P30 CA10815). P.A.J. and R.B. began a sponsored research agreement with Critical Biologics Inc in May 2008 involving measurements of plasma gelsolin levels, but not otherwise related to the present study. This study was established before the SRA was initiated, and no support from it enabled any of the work presented in this manuscript. More than 7 years ago, M.J.DiN. served as a paid consultant to Biogen that was developing aerosolized rhGSN for the treatment of lung disease in patients with cystic fibrosis. He has more recently advised Critical Biologics Corporation (which has acquired the rights to rhGSN) regarding potential diagnostic and therapeutic uses of pGSN on an informal basis and without reimbursement. He is currently an employee of Merck Research Laboratories, which has no past or present ties to the development of gelsolin.
Authors' contribution: E.P.: study design, planning and overseeing of the experiments, data analysis and interpretation, manuscript preparation. M.P., G.R.: technical assistance. R.B.: technical assistance and manuscript preparation. L.A., J.C., P.A.J. and M.J.DiN.: data interpretation and manuscript preparation. J.O. and J.R.K.: patient recruitment. K.C.M.: patient recruitment, data interpretation and manuscript preparation. L.J.M.: study design, data interpretation and manuscript preparation.
Data presented previously at 15th Conference on retroviruses and opportunistic infections, Boston, USA, February 3-February 6 2008, and published as abstract (number 299) in the conference's abstract book (page 162).
1. Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, et al
. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 2006; 12:1365–1371.
2. Hailman E, Lichenstein HS, Wurfel MM, Miller DS, Johnson DA, Kelley M, et al
. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med 1994; 179:269–277.
3. Frey EA, Miller DS, Jahr TG, Sundan A, Bazil V, Espevik T, et al
. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med 1992; 176:1665–1671.
4. Kitchens RL, Thompson PA. Modulatory effects of sCD14 and LBP on LPS-host cell interactions. J Endotoxin Res 2005; 11:225–229.
5. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249:1431–1433.
6. Cohen IR, Norins LC. Natural human antibodies to gram-negative bacteria: immunoglobulins G, A, and M. Science 1966; 152:1257–1259.
7. Strutz F, Heller G, Krasemann K, Krone B, Muller GA. Relationship of antibodies to endotoxin core to mortality in medical patients with sepsis syndrome. Intensive Care Med 1999; 25:435–444.
8. Bucki R, Georges PC, Espinassous Q, Funaki M, Pastore JJ, Chaby R, Janmey PA. Inactivation of endotoxin by human plasma gelsolin. Biochemistry 2005; 44:9590–9597.
9. DiNubile MJ. Plasma gelsolin: in search of its raison d'etre. Focus on ‘Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin’. Am J Physiol Cell Physiol 2007; 292:C1240–C1242.
10. Goetzl EJ, Lee H, Azuma T, Stossel TP, Turck CW, Karliner JS. Gelsolin binding and cellular presentation of lysophosphatidic acid. J Biol Chem 2000; 275:14573–14578.
11. Mounzer KC, Moncure M, Smith YR, Dinubile MJ. Relationship of admission plasma gelsolin levels to clinical outcomes in patients after major trauma. Am J Respir Crit Care Med 1999; 160:1673–1681.
12. Sun HQ, Yamamoto M, Mejillano M, Yin HL. Gelsolin, a multifunctional actin regulatory protein. J Biol Chem 1999; 274:33179–33182.
13. Papasavvas E, Azzoni L, Pistilli M, Hancock A, Reynolds G, Gallo C, et al
. Increased soluble vascular cell adhesion molecule-1 plasma levels and soluble intercellular adhesion molecule-1 during antiretroviral therapy interruption and retention of elevated soluble vascular cellular adhesion molecule-1 levels following resumption of antiretroviral therapy. AIDS 2008; 22:1153–1161.
14. Papasavvas E, Kostman JR, Mounzer K, Grant RM, Gross R, Gallo C, et al
. Randomized, controlled trial of therapy interruption in chronic HIV-1 infection. PLoS Med 2004; 1:e64.
15. Papasavvas E, Kostman JR, Thiel B, Pistilli M, Mackiewicz A, Foulkes A, et al
. HIV-1-specific CD4+ T cell responses in chronically HIV-1 infected blippers on antiretroviral therapy in relation to viral replication following treatment interruption. J Clin Immunol 2006; 26:40–54.
16. Barclay GR. Endogenous endotoxin-core antibody (EndoCAb) as a marker of endotoxin exposure and a prognostic indicator: a review. Prog Clin Biol Res 1995; 392:263–272.
17. Kivilaakso E, Valtonen VV, Malkamaki M, Palmu A, Schroder T, Nikki P, et al
. Endotoxaemia and acute pancreatitis: correlation between the severity of the disease and the antienterobacterial common antigen antibody titre. Gut 1984; 25:1065–1070.
18. Windsor JA, Fearon KC, Ross JA, Barclay GR, Smyth E, Poxton I, et al
. Role of serum endotoxin and antiendotoxin core antibody levels in predicting the development of multiple organ failure in acute pancreatitis. Br J Surg 1993; 80:1042–1046.
19. Titanji K, De Milito A, Cagigi A, Thorstensson R, Grutzmeier S, Atlas A, et al
. Loss of memory B cells impairs maintenance of long-term serologic memory during HIV-1 infection. Blood 2006; 108:1580–1587.
20. Kruger C, Schutt C, Obertacke U, Joka T, Muller FE, Knoller J, et al
. Serum CD14 levels in polytraumatized and severely burned patients. Clin Exp Immunol 1991; 85:297–301.
21. Blanco A, Solis G, Arranz E, Coto GD, Ramos A, Telleria J. Serum levels of CD14 in neonatal sepsis by Gram-positive and Gram-negative bacteria. Acta Paediatr 1996; 85:728–732.
22. Landmann R, Zimmerli W, Sansano S, Link S, Hahn A, Glauser MP, Calandra T. Increased circulating soluble CD14 is associated with high mortality in gram-negative septic shock. J Infect Dis 1995; 171:639–644.
23. Horneff G, Sack U, Kalden JR, Emmrich F, Burmester GR. Reduction of monocyte-macrophage activation markers upon anti-CD4 treatment. Decreased levels of IL-1, IL-6, neopterin and soluble CD14 in patients with rheumatoid arthritis. Clin Exp Immunol 1993; 91:207–213.
24. Nockher WA, Wigand R, Schoeppe W, Scherberich JE. Elevated levels of soluble CD14 in serum of patients with systemic lupus erythematosus. Clin Exp Immunol 1994; 96:15–19.
25. Lien E, Aukrust P, Sundan A, Muller F, Froland SS, Espevik T. Elevated levels of serum-soluble CD14 in human immunodeficiency virus type 1 (HIV-1) infection: correlation to disease progression and clinical events. Blood 1998; 92:2084–2092.
26. Nockher WA, Bergmann L, Scherberich JE. Increased soluble CD14 serum levels and altered CD14 expression of peripheral blood monocytes in HIV-infected patients. Clin Exp Immunol 1994; 98:369–374.
27. Funderburg N, Luciano AA, Jiang W, Rodriguez B, Sieg SF, Lederman MM. Toll-like receptor ligands induce human T cell activation and death, a model for HIV pathogenesis. PLoS ONE 2008; 3:e1915.
28. Meier A, Alter G, Frahm N, Sidhu H, Li B, Bagchi A, et al
. MyD88-dependent immune activation mediated by human immunodeficiency virus type 1-encoded Toll-like receptor ligands. J Virol 2007; 81:8180–8191.