Gut microbiota diversity predicts immune status in HIV-1 infection.

OBJECTIVE: HIV-1 infection is characterized by altered intestinal barrier, gut microbiota dysbiosis, and systemic inflammation. We hypothesized that changes of the gut microbiota predict immune dysfunction and HIV-1 progression, and that antiretroviral therapy (ART) partially restores the microbiota composition. DESIGN: An observational study including 28 viremic patients, three elite controllers, and nine uninfected controls. Blood and stool samples were collected at baseline and for 19 individuals at follow-up (median 10 months) during ART. METHODS: Microbiota composition was determined by 16S rRNA sequencing (Illumina MiSeq). Soluble markers of microbial translocation and monocyte activation were analyzed by Limulus Amebocyte Lysate assay or ELISA. RESULTS: Several alpha-diversity measures, including number of observed bacterial species and Shannon index, were significantly lower in viremic patients compared to controls. The alpha diversity correlated with CD4 T-cell counts and inversely with markers of microbial translocation and monocyte activation. In multivariate linear regression, for every age and sex-adjusted increase in the number of bacterial species, the CD4 T-cell count increased with 0.88 (95% confidence interval 0.35-1.41) cells/µl (P = 0.002). After introduction of ART, microbiota alterations persisted with further reduction in alpha diversity. The microbiota composition at the genus level was profoundly altered in viremic patients, both at baseline and after ART, with Prevotella reduced during ART (P < 0.007). CONCLUSIONS: Gut microbiota alterations are closely associated with immune dysfunction in HIV-1 patients, and these changes persist during short-term ART. Our data implicate that re-shaping the microbiota may be an adjuvant therapy in patients commencing successful ART.

Role of translocated bacterial flagellin in monocyte activation among individuals with chronic HIV-1 infection.

Monocyte activation has been identified as a predictor of mortality and morbidity in HIV-1 infection. This study investigated translocated bacterial flagellin as a potential contributor to systemic monocyte activation via Toll-like receptor 5 (TLR5) stimulation.We demonstrated that circulating flagellin correlated to anti-flagellin, which was associated with soluble markers of microbial translocation (LPS, LBP) and monocyte activation (sCD14, sCD163). Flagellin exposure in vitro reduced monocyte TLR5 expression and the magnitude of reduction was correlated to anti-flagellin levels, indicative of previous flagellin exposure. Circulating anti-flagellin and basal TLR5 expression were both associated with basal and flagellin-stimulated monocyte cytokine production, where HIV + and HIV − differed in their cytokine patterns (IL-1ß, IL-6, IL-8).Our results suggest that translocated flagellin contributes to systemic immune activation in HIV-1 infection and reduces monocyte surface TLR5 expression resulting in a hyperactivated state with elevated basal cytokine production and reduced ability to respond to further TLR5 stimulation.

Effects of Co-Trimoxazole on Microbial Translocation in HIV-1-Infected Patients Initiating Antiretroviral Therapy.

Microbial translocation (MT) contributes to immune activation during HIV-1 infection, and persists after initiation of antiretroviral therapy (ART). We investigated whether levels of MT markers are influenced by the use of co-trimoxazole (TMP-SMX) in HIV-1 patients. Plasma samples were obtained from HIV-1-infected patients initiating ART with (n=13) or without (n=13) TMP-SMX prophylaxis. Markers of MT [lipopolysaccharide-binding protein (LBP), lipopolysaccharide (LPS), soluble CD14 (sCD14), and intestinal fatty acid binding protein (I-FABP)] were assessed at baseline (BL), at 1 month, and at 1 year by the Limulus Amebocyte Lysate Assay or ELISA. BL levels of LBP were elevated in both categories of patients; they were highest in patients starting ART and TMP-SMX (median, µg/ml: 36.7 vs. 4.3, respectively, p=0.001) and correlated inversely with CD4(+) T cell counts (ρ=-0.65; p=0.005). Patients receiving ART and TMP-SMX had a significant reduction in LBP between BL and 1 year (median, µg/ml: 36.7 vs. 11.1; p=0.003). In contrast, levels of LPS at BL were lower in patients starting ART and TMP-SMX compared to those without TMP-SMX (median, pg/ml: 221 vs. 303 respectively; p=0.002) and did not change at 1 year. The increased BL levels of sCD14 had declined in both groups at 1 year. No difference in I-FABP levels was found between BL and 1 year. Concomitant use of ART and TMP-SMX reduces microbial translocation markers LBP and sCD14, probably due to its impact on the gut microbiota. Effective ART for 1 year does not restore gut-blood barrier dysfunction.

Multiparametric bioinformatics distinguish the CD4/CD8 ratio as a suitable laboratory predictor of combined T cell pathogenesis in HIV infection.

HIV disease progression is characterized by numerous pathological changes of the cellular immune system. Still, the CD4 cell count and viral load represent the laboratory parameters that are most commonly used in the clinic to determine the disease progression. In this study, we conducted an interdisciplinary investigation to determine which laboratory parameters (viral load, CD4 count, CD8 count, CD4 %, CD8 %, CD4/CD8) are most strongly associated with pathological changes of the immune system. Multiparametric flow cytometry was used to assess markers of CD4(+) and CD8(+) T cell activation (CD38, HLA-DR), exhaustion (PD-1, Tim-3), senescence (CD28, CD57), and memory differentiation (CD45RO, CD27) in a cohort of 47 untreated HIV-infected individuals. Using bioinformatical methods, we identified 139 unique populations, representing the "combined T cell pathogenesis," which significantly differed between the HIV-infected individuals and healthy control subjects. CD38, HLA-DR, and PD-1 were particularly expressed within these unique T cell populations. The CD4/CD8 ratio was correlated with more pathological T cell populations (n = 10) and had a significantly higher average correlation coefficient than any other laboratory parameters. We also reduced the dimensionalities of the 139-unique populations by Z-transformations and principal component analysis, which still identified the CD4/CD8 ratio as the preeminent surrogate of combined T cell pathogenesis. Importantly, the CD4/CD8 ratio at baseline was shown to be significantly associated with CD4 recovery 2 y after therapy initiation. These results indicate that the CD4/CD8 ratio would be a suitable laboratory predictor in future clinical and therapeutic settings to monitor pathological T cell events in HIV infection.

Pattern of microbial translocation in patients living with HIV-1 from Vietnam, Ethiopia and Sweden.

INTRODUCTION: The role of microbial translocation (MT) in HIV patients living with HIV from low- and middle-income countries (LMICs) is not fully known. The aim of this study is to investigate and compare the patterns of MT in patients from Vietnam, Ethiopia and Sweden. METHODS: Cross-sectional samples were obtained from treatment-naïve patients living with HIV-1 and healthy controls from Vietnam (n=83; n=46), Ethiopia (n=9492; n=50) and Sweden (n=51; n=19). Longitudinal samples were obtained from a subset of the Vietnamese (n=24) in whom antiretroviral therapy (ART) and tuberculostatics were given. Plasma lipopolysaccharide (LPS), sCD14 and anti-flagellin IgG were determined by the endpoint chromogenic Limulus Amebocyte Assay and enzyme-linked immunosorbent assay. RESULTS: All three biomarkers were significantly increased in patients living with HIV-1 from all countries as compared to controls. No differences were found between males and females. Vietnamese and Ethiopian patients had significantly higher levels of anti-flagellin IgG and LPS, as compared to Swedes. ART reduced these levels for the Vietnamese. Vietnamese patients given tuberculostatics at initiation of ART had significantly lower levels of anti-flagellin IgG and higher sCD14. The biomarkers were lower in Vietnamese who did not develop opportunistic infection. CONCLUSIONS: Higher MT is common in patients living with HIV compared to healthy individuals, and in patients from LMICs compared to patients from a high-income country. Treatment with tuberculostatics decreased MT while higher levels of MT are associated with a poorer clinical outcome.

Microbial translocation contribute to febrile episodes in adults with chemotherapy-induced neutropenia.

In this study we sought to determine the contribution of microbial translocation to febrile episodes with no attributable microbiological cause (Fever of Unknown Origin, FUO) in an adult febrile neutropaenic cohort. Endotoxin concentrations were measured with the chromogenic Limulus Amoebocyte Assay and used as a direct measure of bacterial products whilst soluble CD14 (sCD14), measured with ELISA was selected as an indicator of the early host response to endotoxins. Endotoxin concentrations in this cohort were generally elevated but did not differ with the presentation of fever. Further stratification of the febrile episodes based on the microbiological findings revealed significantly (p = 0.0077) elevated endotoxin concentrations in FUO episodes compared with episodes with documented bacterial and viral findings. sCD14 concentrations were however, elevated in febrile episodes (p = 0.0066) and no association was observed between sCD14 concentration and microbiological findings. However, FUO episodes and episodes with Gram-negative bacteraemia were associated with higher median sCD14 concentrations than episodes with Gram-positive bacteraemia (p = 0.030). In conclusion, our findings suggest that in the absence of microbiological findings, microbial translocation could contribute to febrile episodes in an adult neutropaenic cohort. We further observed an association between prophylactic antibiotic use and increased plasma endotoxin concentrations (p = 0.0212).

Kinetics of microbial translocation markers in patients on efavirenz or lopinavir/r based antiretroviral therapy.

OBJECTIVES: We investigated whether there are differences in the effects on microbial translocation (MT) and enterocyte damage by different antiretroviral therapy (ART) regimens after 1.5 years and whether antibiotic use has impact on MT. In a randomized clinical trial (NCT01445223) on first line ART, patients started either lopinavir/r (LPV/r) (n = 34) or efavirenz (EFV) containing ART (n = 37). Lipopolysaccharide (LPS), sCD14, anti-flagellin antibodies and intestinal fatty acid binding protein (I-FABP) levels were determined in plasma at baseline (BL) and week 72 (w72). RESULTS: The levels of LPS and sCD14 were reduced from BL to w72 (157.5 pg/ml vs. 140.0 pg/ml, p = 0.0003; 3.13 ug/ml vs. 2.85 ug/ml, p = 0.005, respectively). The levels of anti-flagellin antibodies had decreased at w72 (0.35 vs 0.31 [OD]; p<0.0004), although significantly only in the LPV/r arm. I-FABP levels increased at w72 (2.26 ng/ml vs 3.13 ng/ml; p<0.0001), although significantly in EFV treated patients only. Patients given antibiotics at BL had lower sCD14 levels at w72 as revealed by ANCOVA compared to those who did not receive (Δ = -0.47 µg/ml; p = 0.015). CONCLUSIONS: Markers of MT and enterocyte damage are elevated in untreated HIV-1 infected patients. Long-term ART reduces the levels, except for I-FABP which role as a marker of MT is questionable in ART-experienced patients. Why the enterocyte damage seems to persist remains to be established. Also antibiotic usage may influence the kinetics of the markers of MT. TRIAL REGISTRATION: ClinicalTrials.gov NCT01445223.

Circulating levels of HMGB1 are correlated strongly with MD2 in HIV-infection: possible implication for TLR4-signalling and chronic immune activation.

Progressive HIV infection is characterized by profound enterocyte damage, microbial translocation and chronic immune activation. We aimed to test whether High Mobility Group Box protein 1(HMGB1), a marker of cell death, alone, or in combination with LPS, might contribute to HIV-associated immune activation and progression. Altogether, 29 untreated HIV-infected individuals, 25 inflammatory bowel disease (IBD) patients and 30 controls were included. HIV-infected patients had lower plasma LPS levels than IBD patients, but higher levels of soluble CD14 and Myeloid Differentiation (MD) 2, which interacts with TLR4 to initiate LPS-signalling. Furthermore, plasma levels of HMGB1 and MD2 were correlated directly within the HIV-infected cohort (r = 0.89, P < 0.001) and the IBD-cohort (r = 0.85, P < 0.001), implying HMGB1 signalling through the MD2/TLR4-pathway. HMGB1 and LPS, although not inter-correlated, were both moderately (r = 0.4) correlated with CD38 density on CD8+ T cells in HIV progressors. The highest levels of CD38 density and MD2 were found in progressors with plasma levels of both LPS and HMGB1 above the fiftieth percentile. Our results could imply that, in some patients, immune activation is triggered by microbial translocation, in some by cell death and in some by HMGB1 in complex with bacterial products through activation of the MD2/TLR4-pathway.

Implications of the release of high-mobility group box 1 protein from dying cells during human immunodeficiency virus type 1 infection in vitro.

Plasma levels of high-mobility group box 1 protein (HMGB1) are elevated during the course of human immunodeficiency virus type 1 (HIV-1) infection and the molecule has an impact on virus replication. This study investigated the mode of cell death and release of HMGB1 during HIV-1 infection in vitro. MT4 cells and primary CD4(+) T cells were infected with HIV-1 isolates, and HMGB1 release was monitored in relation to cytopathic effects (CPE) and apoptosis. HMGB1 release from cells was analysed by Western blotting. For MT4 cells, an enzyme-linked immunosorbent spot (ELISPOT) assay was adapted to measure the release during necrosis. Lactate dehydrogenase (LDH) activity was quantified using a commercial assay. Flow cytometry was used to determine the level of infection and apoptosis. MT4 cells were > or =90 % infected at 48 h post-infection (p.i.). CPE was first observed at 60 h and correlated with release of HMGB1, LDH activity and caspase-3 (C3) activation. HMGB1 spots were clearly detected by ELISPOT assay at 72 h p.i. Annexin V and C3 staining showed that apoptosis was substantially involved in HIV-1-related cell death. Addition of Z-VAD (a caspase inhibitor) in a single dose at 24 or 40 h p.i. decreased both the number of caspase-positive cells and the release of HMGB1. Infection of primary CD4(+) T cells showed a 22 % (median) infection rate at 96 h. Related CPE corresponded to LDH and HMGB1 release. Both necrosis and apoptosis contributed to HMGB1 liberation during HIV-1-induced cell death and the protein could induce tumour necrosis factor-alpha release from peripheral mononuclear blood cells. These data imply that passive HMGB1 release contributes to the excessive immune activation characteristic of HIV-1 pathogenesis.

Elevated plasma levels of high mobility group box protein 1 in patients with HIV-1 infection.

High mobility group box protein 1 (HMGB1) is a potent proinflammatory mediator. It has a dichotomic effect on HIV-1 replication in vitro but its role in vivo is unknown. Here we report the novel finding that plasma HMGB1 levels are elevated in HIV-1-infected patients, with the highest concentrations in patients with clinical complications. HMGB1 is likely to contribute to immunoactivation in HIV-1 infection in vivo.

HMGB1 activates replication of latent HIV-1 in a monocytic cell-line, but inhibits HIV-1 replication in primary macrophages.

High mobility group box protein 1 (HMGB1) is an abundant component of mammalian cells that can be released into extracellular milieu actively or by cells that undergo necrosis. Exposure of inflammatory and endothelial cells to HMGB1 leads to the release of cytokines, including TNF-alpha and IL-6. To evaluate the impact of exogenous HMGB1 on viral replication in HIV-1 infected cells, we studied models of latent and acute infection. Extracellular HMGB1 dose dependently increased HIV-1 replication in the monocytic cells, U1, which is an established model for studying latent HIV-1 infection. Dexamethasone, a known inhibitor of NF-kappaB signaling in U1 cells, inhibited HMGB1-induced stimulation of the viral production. Addition of HMGB1 to primary monocytic cells with active HIV-1 infection elicited the opposite effect, due to suppression of the viral replication. The mechanism of this unexpected finding was explained by an HMGB1-mediated increased release of chemokines (RANTES, MIP-1alpha, and MIP-1beta) that are known to inhibit HIV-1 replication. The stimulatory effect of the HMGB1 was not present when latently infected T-cells (ACH-2) were used as target cells. Our data suggest that extracellular HMGB1 has a dichotomic effect on the HIV-1 infection in monocytes but not in lymphocytes. Both activation of latent HIV-1 infection and inhibition of active replication can thus be seen in vitro.

Early induction of leukemia inhibitor factor (LIF) in acute HIV-1 infection.

BACKGROUND: Leukemia inhibitor factor (LIF) is thought to play a substantial role in protecting CD4 T cells in lymphoid tissues (LT) from infection by HIV-1. OBJECTIVE: To investigate whether primary HIV-1 infected subjects with sustained virological control (< 1000 HIV-1 RNA copies/ml plasma) post-cessation antiretroviral therapy (ART) had a higher initial LIF response during primary HIV-1 infection (PHI) as compared with those individuals who did not achieve a similar control (> 9000 HIV-1 RNA copies/ml plasma) of HIV-1 replication. MATERIAL AND METHODS: Consecutively obtained HIV plasma samples were collected from primary HIV-1 infected subjects. A group of acutely Epstein-Barr virus-infected subjects and a group of HIV-1-seronegative healthy individuals served as controls. All samples were tested by ELISA for LIF and sgp130, the soluble form of LIF's signalling receptor. RESULTS: LIF and sgp130 plasma levels were significantly increased in primary HIV-1-infected subjects as compared with HIV-1-seronegative controls. Peak plasma levels of LIF occurred during the first week of PHI whereas sgp130 peaked between 2 and 4 weeks after the onset of PHI. Furthermore a positive correlation was found between viral load and plasma levels of LIF during PHI. Both LIF and sgp130 plasma concentrations were significantly lower during the viral rebound phase after treatment interruption as compared with the PHI phase. CONCLUSIONS: LIF induction occurred in the initial stages of viral dissemination during PHI. It may be a part of the virally induced generalized pro-inflammatory response. LIF levels at PHI did not predict low levels of HIV viraemia after discontinuation of ART. LIF was not increased following ART interruption in this early treated population.