Evidence for Polymicrobic Flora Translocating in Peripheral Blood of HIV-Infected Patients with Poor Immune Response to Antiretroviral Therapy

In advanced HIV infection, the homeostatic balance between gastrointestinal indigenous bacteria and gut immunity fails and microbes are able to overcome the intestinal barrier and gain the systemic circulation. Because microbial translocation is not fully controlled by antiviral therapy and is associated with inefficient CD4+ reconstitution, we investigated the profile of translocating bacteria in peripheral blood of 44 HIV-infected patients starting therapy with advanced CD4+ T-lymphopenia and displaying poor CD4+ recovery on virologically suppressive HAART. According to CD4+ reconstitution at 12-months HAART, patients were considered Partial Immunological Responders, PIRs (CD4+≥250/µl, n = 29) and Immunological non Responders, INRs (CD4+<200/µl, n = 15)). We show that PIRs and INRs present similarly elevated plasma levels of lipopolysaccharide (LPS) and its ligand sCD14 that were not lowered by virologically suppressive therapy. Bacterial 16S rRNA gene amplification and sequencing resulted in a highly polymicrobic peripheral blood microbiota both prior and after 12-month HAART. Several differences in bacterial composition were shown between patients' groups, mainly the lack of probiotic Lactobacillaceae both prior and after therapy in INRs. Failure to control microbial translocation on HAART is associated with a polymicrobic flora circulating in peripheral blood that is not substantially modified by therapy

Increased Bone Marrow Interleukin-7 (IL-7)/IL-7R Levels but Reduced IL-7 Responsiveness in HIV-Positive Patients Lacking CD4+ Gain on Antiviral Therapy

Background: The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Ra in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ #200/ml) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA#50), 12 complete failures (CFs; HIV-RNA.1000), and 23 HIVseronegative subjects. Methods: We studied plasma IL-7 levels, IL-7Ra+CD4+/CD8+ T-cell proportions, IL-7Ra mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Ra mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells. Results: Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Ra CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Ra mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04). Conclusions: Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Ra expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor

Circulating sCD14 Is Associated with Virological Response to Pegylated-Interferon-Alpha/Ribavirin Treatment in HIV/HCV Co-Infected Patients

Microbial translocation (MT) through the gut accounts for immune activation and CD4+ loss in HIV and may influence HCV disease progression in HIV/HCV co-infection. We asked whether increased MT and immune activation may hamper anti-HCV response in HIV/HCV patients.98 HIV/HCV patients who received pegylated-alpha-interferon (peg-INF-alpha)/ribavirin were retrospectively analyzed. Baseline MT (lipopolysaccharide, LPS), host response to MT (sCD14), CD38+HLA-DR+CD4+/CD8+, HCV genotype, severity of liver disease were assessed according to Early Virological Response (EVR: HCV-RNA <50 IU/mL at week 12 of therapy or ≥2 log(10) reduction from baseline after 12 weeks of therapy) and Sustained Virological Response (SVR: HCV-RNA <50 IU/mL 24 weeks after end of therapy). Mann-Whitney/Chi-square test and Pearson's correlation were used. Multivariable regression was performed to determine factors associated with EVR/SVR.71 patients displayed EVR; 41 SVR. Patients with HCV genotypes 1-4 and cirrhosis presented a trend to higher sCD14, compared to patients with genotypes 2-3 (p = 0.053) and no cirrhosis (p = 0.052). EVR and SVR patients showed lower levels of circulating sCD14 (p = 0.0001, p = 0.026, respectively), but similar T-cell activation compared to Non-EVR (Null Responders, NR) and Non-SVR (N-SVR) subjects. sCD14 resulted the main predictive factor of EVR (0.145 for each sCD14 unit more, 95%CI 0.031-0.688, p = 0.015). SVR was associated only with HCV genotypes 2-3 (AOR 0.022 for genotypes 1-4 vs 2-3, 95%CI 0.001-0.469, p = 0.014).In HIV/HCV patients sCD14 correlates with the severity of liver disease and predicts early response to peg-INF-alpha/ribavirin, suggesting MT-driven immune activation as pathway of HIV/HCV co-infection and response to therapy

Patients' characteristics.

<p><b>NOTE:</b> Data are median (IQR -Interquartile range-). FRs: Full Responders; INRs : Immunological Non Responders; IDU: intravenous drug user; HAART: highly active antiretroviral therapy; NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor; PI: protease inhibitor.</p>a<p>p<.01 for FRs vs INRs;</p>b<p>p<.01 for T0 vs T12.</p

Characterization of translocating microflora in HIV+ antiretroviral naive patients.

<p>Patients' plasma samples were examined before and after 12 months of HAART for the presence and identification of DNA bacterial fragments using a broad-range 16S rRNA gene PCR amplification followed by sequencing analysis. At baseline, 14/44 (32%) HIV+ patients yielded a positive PCR amplification, whereas at T12, 7/44 (16%) HIV+ patients were PCR-positive. Sequencing analysis of HIV positive patients as a whole revealed multiple bacterial orders for each patient with no differences in the bacterial composition between baseline and T12. Positive (%) = # patients displaying a specific bacterial order/total # of PCR positive patients. Negative (%) = # patients negative for a specific bacterial order/total # of PCR positive patients.</p

Identification of bacterial families in PIRs and INRs.

<p>Positive (%) = # patients displaying a specific bacterial family/total # of PCR positive PIRs (or INRs). Negative (%) = # patients negative for a specific bacterial family/total # of PCR positive PIRs (or INRs). PIRs and INRs presented a different profile in term of bacteria families. (<b>A</b>) PIRs displayed a similar composition of bacterial families between baseline and T12. (<b>B</b>) No major changes in bacterial families were seen in INRs after 12 months-HAART. At baseline, the significant differences between the two groups concerned <i>Lactobacillaceae</i> *(PIRs 3/6, 50% vs INRs 0/8 0%; p = .05) and <i>Pseudomonadaceae *</i>(PIRs 5/6, 83% vs INRs 1/8, 13%; p = .026). No differences between PIRs and INRs at T12.</p

Activated HLA-DR+CD4+ and CD8+ T-cells according to liver fibrosis, cirrhosis and HCV genotypes.

<p><b>a</b>)<b>-b</b>) Activated HLA-DR+CD4+ and CD8+ T-cells were compared between patients with advanced fibrosis (AF) and non advanced fibrosis (N-AF). <b>c</b>)<b>-d</b>) Activated HLA-DR+CD4+ and CD8+ T-cells were compared between patients with cirrhosis and absence of cirrhosis (N-Cirrhosis). <b>e</b>)<b>-f</b>) Activated HLA-DR+CD4+ and CD8+ T-cells were compared between patients with HCV genotypes 1–4 and genotypes 2–3. Each point represents the value from one subject's plasma. Activated HLA-DR+CD4+ and CD8+ T-cells % values are presented. AF = advanced fibrosis – N-AF = non advanced fibrosis. p-values were assessed by Mann Whitney U test. p>0.05 was considered non significant (NS).</p

Baseline demographic and immuno-virological characteristics of study population.

<p><b>LEGEND.</b> Data are presented as *median, (IQR) and °absolute number, (%). Differences between groups were compared by *Mann Whitney U test and °χ2 test. EVR, Early Virological Response: undetectable serum HCV-RNA (<50 IU/mL) or ≥2 log₁₀ reduction from baseline after 12 weeks of therapy; NR, Null Responders: serum HCV-RNA ≥50 IU/mL and <2 log₁₀ reduction from baseline. cART, Combination Antiretroviral therapy; NRTI, Nucleoside Reverse Transcriptase Inhibitors; NNRTI, Non Nucleoside Reverse Transcriptase Inhibitors; PI, Protease Inhibitors. MSM, men who have sex with men; WSM, women who have sex with men; IDUs, injection drug users. HCV, hepatitis C virus; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen. AST, aspartate aminotransferase; ALT, alanine aminotransferase. BMI, Body Mass Index. HOMA index, Homeostatic Model Assessment index.</p

Association between markers of microbial translocation and Early Virological Response to anti-HCV treatment.

<p><b>LEGEND.</b> LPS, soluble CD14, CD4+ T cells/µL, age, HCV-RNA log₁₀ cp/mL for each unit more. sCD14 and LPS were measured in plasma samples; sCD14 µg/mL, LPS pg/mL. Multivariate analysis was performed in 65/98 patients for whom all the clinical, epidemiological and biological parameters under study were available.</p><p>OR, odds ratio; AOR, adjusted odds ratio; CI, confidence interval. p>0.05 was considered non significant.</p