Ricardo Khouri

Heme Impairs Prostaglandin E2 and TGF-β Production by Human Mononuclear Cells via Cu/Zn Superoxide Dismutase: Insight into the Pathogenesis of Severe Malaria

In many hemolytic disorders, such as malaria, the release of free heme has been involved in the triggering of oxidative stress and tissue damage. Patients presenting with severe forms of malaria commonly have impaired regulatory responses. Although intriguing, there is scarce data about the involvement of heme on the regulation of immune responses. In this study, we investigated the relation of free heme and the suppression of anti-inflammatory mediators such as PGE2 and TGF-β in human vivax malaria. Patients with severe disease presented higher hemolysis and higher plasma concentrations of Cu/Zn superoxide dismutase (SOD-1) and lower concentrations of PGE2 and TGF-β than those with mild disease. In addition, there was a positive correlation between SOD-1 concentrations and plasma levels of TNF-α. During antimalaria treatment, the concentrations of plasma SOD-1 reduced whereas PGE2 and TGF-β increased in the individuals severely ill. Using an in vitro model with human mononuclear cells, we demonstrated that the heme effect on the impairment of the production of PGE2 and TGF-β partially involves heme binding to CD14 and depends on the production of SOD-1. Aside from furthering the current knowledge about the pathogenesis of vivax malaria, the present results may represent a general mechanism for hemolytic diseases and could be useful for future studies of therapeutic approaches.

IFN-β induces greater antiproliferative and proapoptotic effects and increased p53 signaling compared with IFN-α in PBMCs of Adult T-cell Leukemia/Lymphoma patients

Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, to AB, JVW and AMV “PVE”), PRONEX (CNPq-FAPESB to AB and JVW and LF), FWO (Grant: ZKC1280-00-W10 and G0D6817N to AMV)

Superior antiviral and antiproliferative activity of IFN-beta vs. IFN-alpha in primary ATL cells occurs downstream of STAT1 signaling

Adult T-cell leukemia (ATL) is an aggressive CD4+CD25+ leukemia with poor prognosis, which usually develops several decades after HTLV-1 infection. In contrast to HIV-infection, the treatment of HTLV-1-associated diseases rely on a limited number of drugs. For ATL, combination therapy with IFN-alpha+AZT has shown clinical benefit in the non-lymphoma subtypes. Type I IFNs (IFN-alpha/beta) are essential cytokines with proved anti-cancer and antiviral action in vitro and in vivo. Nonetheless, their mechanisms of action in HTLV-1 infection remain unclear and a side-by-side comparison of both type I IFNs has not been performed in ATL. We show, in short-term culture of primary mononuclear cells from ATL patients, that both IFNs cause increased apoptosis, exert an anti-proliferative and antiviral effect, and decrease pro-inflammatory cytokine levels. However, IFN-beta treatment was significantly more effective in inhibiting viral p19 protein levels and lymphoproliferation, as compared to IFN-alpha. This pronounced effect of IFN-beta was explained by an induction of a higher number of known IFN-stimulated genes and antiviral genes by microarray analysis (76 vs. 26 genes were selected with p 2-fold difference vs. control). In PBMCs from healthy donors, ATL patients as well as in HTLV-1-infected cell lines, both IFNs have comparable activity in phosphorylating STATs 1 through 5 (PhosFlow), although phospho-STAT1 levels were up to tenfold higher than phospho-STAT2 through 5. This predominant STAT1-mediated antiviral gene signature was confirmed by Ingenuity Pathway analysis. In conclusion, our data suggest the superior antiviral and antiproliferative activity of IFN-beta vs. IFN-alpha occurs downstream of STAT1 signaling.

A Bayesian network approach to study host and viral genetic correlates of HIV-1 disease progression

Background HIV disease progression is very variable among infected patients. Using classical statistical methods based on a selected number of markers, Casado et al [1] identified a number of host and viral genetic correlates for the clinical definitions of HIV-1 disease progression: elite controllers, long term non progressors including viremic controllers and clinical non progressors, regular progressors and rapid progressors.

Comprehensive antiretroviral restriction factor profiling reveals the evolutionary imprint of the ex vivo and in vivo IFN-β response in HTLV-1-associated neuroinflammation

HTLV-1-Associated Myelopathy (HAM/TSP) is a progressive neuroinflammatory disorder for which no disease-modifying treatment exists. Modest clinical benefit from type I interferons (IFN-α/β) in HAM/TSP contrasts with its recently identified IFN-inducible gene signature. In addition, IFN-α treatment in vivo decreases proviral load and immune activation in HAM/TSP, whereas IFN-β therapy decreases tax mRNA and lymphoproliferation. We hypothesize this “IFN paradox” in HAM/TSP might be explained by both cell type- and gene-specific effects of type I IFN in HTLV-1-associated pathogenesis. Therefore, we analyzed ex vivo transcriptomes of CD4+ T cells, PBMCs and whole blood in healthy controls, HTLV-1-infected individuals, and HAM/TSP patients. First, we used a targeted approach, simultaneously quantifying HTLV-1 mRNA (HBZ, Tax), proviral load and 42 host genes with known antiretroviral (anti-HIV) activity in purified CD4+ T cells. This revealed two major clusters (“antiviral/protective” vs. “proviral/deleterious”), as evidenced by significant negative (TRIM5/TRIM22/BST2) vs. positive correlation (ISG15/PAF1/CDKN1A) with HTLV-1 viral markers and clinical status. Surprisingly, we found a significant inversion of antiretroviral activity of host restriction factors, as evidenced by opposite correlation to in vivo HIV-1 vs. HTLV-1 RNA levels. The anti-HTLV-1 effect of antiviral cluster genes was significantly correlated to their adaptive chimp/human evolution score, for both Tax mRNA and PVL. Six genes of the proposed antiviral cluster underwent lentivirus-driven purifying selection during primate evolution (TRIM5/TRIM22/BST2/APOBEC3F-G-H), underscoring the cross-retroviral evolutionary imprint. Secondly, we examined the genome-wide type I IFN response in HAM/TSP patients, following short-term ex vivo culture of PBMCs with either IFN-α or IFN-β. Microarray analysis evidenced 12 antiretroviral genes (including TRIM5α/TRIM22/BST2) were significantly up-regulated by IFN-β, but not IFN-α, in HAM/TSP. This was paralleled by a significant decrease in lymphoproliferation by IFN-β, but not IFN-α treatment. Finally, using published ex vivo whole blood transcriptomic data of independent cohorts, we validated the significant positive correlation between TRIM5, TRIM22, and BST2 in HTLV-1-infected individuals and HAM/TSP patients, which was independent of the HAM/TSP disease signature. In conclusion, our results provide ex vivo mechanistic evidence for the observed immunovirological effect of in vivo IFN-β treatment in HAM/TSP, reconcile an apparent IFN paradox in HTLV-1 research and identify biomarkers/targets for a precision medicine approach.

B cell costimulatory molecules as potential biomarkers in HAM/TSP

Current therapy in HAM/TSP includes antiretrovirals and immunomodulators, such as corticosteroids and interferons, with modest clinical benefit. Since there are no validated biomarkers to monitor disease severity (DS), progression or prognosis, we investigated the ex vivo expression of CD80 and CD86 as possible biomarkers, as well as the effect of IFN-α/beta on their in vitro expression in 16 Brazilian HAM/TSP patients and 15 healthy controls (HC). Flow cytometry quantification indicated significantly elevated expression of both CD80 and CD86 ex vivo in T (p=0.044 and p=0.0016) and B (p=0.0003 and p=0.0057) cells, as compared to HC. In B cells, the ex vivo percentage of CD80+ positively correlated to DS (p=0.0083, r=0.78), but not age or disease duration, while ex vivo expression of CD80 was gender-biased, being significantly higher in women (p=0.028). Upon in vitro culture, CD80+ (p=0.037) and CD86+ lymphocytes significantly expand (p<0.0001), as compared to ex vivo levels, in patients only. In vitro treatment of patients’ PBMCs with IFN-beta, but not IFN-α resulted in significant stimulation of B cell CD86 expression (p<0.05). In HC, both IFN-α and IFN-beta brought about a significant increase in CD86 expression (p<0.05 and p<0.001). Neither IFN-α nor IFN-beta was able to significantly induce B cell CD80 expression in HC or patients. Consequently, B cell CD86/CD80 ratio was significantly increased upon stimulation with IFN-beta in both HC and patients. In conclusion, we propose novel biomarkers for possible clinical use in HAM/TSP trials: ex vivo CD80+ B cells positively correlating to DS and CD86+ B cells preferentially induced by IFN-beta.