Fareed Ahmad

miR-21 promotes fibrosis in an acute cardiac allograft transplantation model

AIMS Cardiac transplantation is the only curative therapy for end-stage heart failure. Fibrosis is one of the major causes for impaired function of cardiac allografts. MicroRNAs, a class of small non-coding RNAs, play a critical role in the development of cardiovascular disease, but the role of microRNAs in cardiac allograft failure is not well understood. METHODS AND RESULTS To uncover a role of microRNAs during cardiac graft fibrosis, we generated global microRNA profiles in allogeneic (BALB/c in C57BL/6N) and isogeneic (C57BL/6N in C57BL/6N) murine hearts after transplantation. miR-21 together with cardiac fibrosis was increased in cardiac allografts compared with isografts. Likewise, patients with cardiac rejection after heart transplantation showed increased cardiac miR-21 levels. miR-21 was induced upon treatment with IL-6 in a monocyte cell line. Overexpression of miR-21 in this monocyte cell line activated a fibrotic gene programme and promoted monocyte-to-fibrocyte transition together with activation of chemokine (C-C) motif ligand 2 (monocyte chemoattractant protein 1) via the phosphatase and tensin homologue/activator protein 1 regulatory axis. In vivo, both genetic and pharmacological inhibition of miR-21 successfully reduced fibrosis and fibrocyte accumulation in cardiac allografts. CONCLUSION Thus, inhibition of miR-21 is a novel strategy to target fibrosis development in cardiac allografts.

Phenotypically and functionally distinct subsets contribute to the expansion of CD56-/CD16+ natural killer cells in HIV infection.

Objective:Chronic HIV infection has been associated with activation and increased turnover of natural killer (NK) cells as well as with disturbed homeostasis of the NK cell compartment, including loss of CD56+ NK cells and accumulation of dysfunctional CD56−/CD16+ NK cells. We performed a comprehensive phenotypical and functional characterization of this population. Design:A cross-sectional study was performed to analyze CD56−/CD16+ NK cells from 34 untreated HIV-infected and 15 seronegative individuals. Methods:NK cells were analyzed by flow cytometry. Degranulation was assessed by measuring their expression of CD107a after stimulation with K562 cells, interleukin-12 and interleukin-15. Results:CD56−/CD16+ NK cells are heterogeneous and composed of two populations, namely CD122−/CCR7+ cells and CD122+/CCR7− cells. We show that expanded CD122+ but not CCR7+ cells in HIV-seropositive individuals are characterized by expression of senescence marker CD57 similarly to CD56dim/CD16+ NK cells along with expression of KIRs, CD8, perforin and granzyme B. Despite expression of perforin and granzyme B, CD57 expressing cells exhibited less numbers of degranulating cells as measured by CD107a, indicating their functional impairment. However, there was no correlation between expansion of total CD56−/CD16+ NK cells or the distinct subpopulations and viral load or CD4 cell count. Conclusion:These data indicate that expansion of CD56−/CD16+ cells in HIV infection is driven by a distinct subset within this population with high expression of terminal differentiation marker with a phenotype resembling CD56dim/CD16+ NK cells.

Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles.

Objective:HIV-1 Vpu and Nef proteins downregulate cell surface levels of natural killer (NK) cell ligands but functional consequences of individual downregulation events are unclear. We tested how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients. Methods:Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors. Results:Cell surface downregulation of CD4, CD317/tetherin, and major histocompatibility complex class 1 that exert biological functions other than NK cell activation were well conserved among patient-derived Vpu and Nef variants. Among NK cell ligands, NK-T-B-antigen, poliovirus receptor, and UL16-binding protein were identified as main targets for Vpu and Nef, the downregulation of which by at least 1 viral protein was highly conserved. NK cell ligands displayed specific sensitivity to Vpu (NK-T-B-antigen) or Nef (poliovirus receptor), and downregulation of cell surface UL16-binding protein was identified as a novel and highly conserved activity of HIV-1 Vpu but not Nef. Conclusions:The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection.

Natural killer cell heterogeneity: cellular dysfunction and significance in HIV-1 immuno-pathogenesis

Natural killer (NK) cells are innate immune effectors that provide first line of defence against viruses. Human NK cells are heterogeneous in nature, and their functions rely on a dynamic balance between germ-line-encoded activating and inhibitory receptors. HIV-1 infection results in altered NK cell receptor repertoire and impaired effector functions including the ability to lyse virus-infected cells and secretion of antiviral cytokine IFN-γ. Over the last decade, additional NK cell subset-specific molecules have been identified, leading to emergence of a more complex cellular diversity than previously thought. Herein, we discuss NK cell subset redistribution, altered receptor repertoire and influence of interaction of polymorphic leucocyte antigen (HLA) and killer cell immunoglobulin-like receptors (KIR) on HIV-1 disease progression.

Negative Checkpoint Regulatory Molecule 2B4 (CD244) Upregulation Is Associated with Invariant Natural Killer T Cell Alterations and Human Immunodeficiency Virus Disease Progression

The CD1d-restricted invariant natural killer T (iNKT) cells are implicated in innate immune responses against human immunodeficiency virus (HIV). However, the determinants of cellular dysfunction across the iNKT cells subsets are seldom defined in HIV disease. Herein, we provide evidence for the involvement of the negative checkpoint regulator (NCR) 2B4 in iNKT cell alteration in a well-defined cohort of HIV-seropositive anti-retroviral therapy (ART) naïve, ART-treated, and elite controllers (ECs). We report on exaggerated 2B4 expression on iNKT cells of HIV-infected treatment-naïve individuals. In sharp contrast to CD4−iNKT cells, 2B4 expression was significantly higher on CD4+ iNKT cell subset. Notably, an increased level of 2B4 on iNKT cells was strongly correlated with parameters associated with HIV disease progression. Further, iNKT cells from ART-naïve individuals were defective in their ability to produce intracellular IFN-γ. Together, our results suggest that the levels of 2B4 expression and the downstream co-inhibitory signaling events may contribute to impaired iNKT cell responses.

Limitation of Simultaneous Analysis of T-Cell Receptor and κ-Deleting Recombination Excision Circles Based on Multiplex Real-Time Polymerase Chain Reaction in Common Variable Immunodeficiency Patients.

Aim of Study: We used a triplex real-time polymerase chain reaction (PCR) to classify our common variable immunodeficiency (CVID) patients into distinct groups according to the amount of their T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs). Materials and Methods: TREC and KREC analysis was performed using a multiplex real-time PCR assay. The T- and B-lymphocyte subsets were measured by flow cytometry. Results: The copy number of TRECs and KRECs was significantly reduced in CVID patients compared to healthy controls. The TREC copy number was inversely correlated with age in both healthy subjects and patients; however, the KREC copy number was inversely correlated with age only in CVID patients. Moreover, no association was seen between TREC/KREC copy number and clinical manifestations such as bronchiectasis, splenomegaly, granulomata, autoimmune cytopenias, organ-specific autoimmunity, enteropathy and lymphoid hyperplasia. Conclusion: TREC and KREC quantification might be a useful tool to differentiate between CVID and combined immunodeficiency, but considering the results of this study a classification of CVID patients in certain groups is hardly possible.

IL-1β limits the extent of human 6-sulfo LacNAc dendritic cell (slanDC)-mediated NK cell activation and regulates CD95-induced apoptosis.

To function optimally, human blood natural killer (NK) cells need to communicate with other immune cells. Previously, it has been shown that NK cells communicate with 6-sulfo LacNAc dendritic cells (slanDCs), which are able to stimulate NK cells in vitro. In this study, we investigated how slanDCs regulate the level of NK cell activation. The secretion of interleukin (IL)-1β by slanDCs during coculture with NK cells increased as a result of signaling via intercellular adhesion molecule-1 on slanDCs following its interaction with lymphocyte function-associated antigen-1 on NK cells. IL-1β induced the expression of Fas receptor (CD95) on NK cells. The binding of Fas ligand (CD178) to CD95 induced the apoptosis of activated NK cells. Moreover, IL-1β also induced increased cyclooxygenase-2 expression in slanDCs, which in turn enabled the cells to secrete prostaglandin (PG)-E2. Consequently, PGE2 acted as a suppressing agent, tuning down the activation level of NK cells. In summary, IL-1β limits the level of NK cell activation by inducing apoptosis and suppression as a homeostatic regulatory function.

Evidence of inflammasome activation and formation of monocyte-derived apoptosis-associated speck-like protein containing a caspase-recruitment domain specks in HIV-1 positive patients

Objective: The formation of large intracellular protein aggregates of the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain; also know as PYCARD) is a hallmark of inflammasome activation. ASC speck-forming cells release the highly proinflammatory cytokine IL-1&bgr; in addition to ASC specks into the extracellular space during pyroptotic cell death. There ASC specks can propagate inflammation to other nonactivated cells or tissues. HIV-1 retroviral infection triggers inflammasome activation of abortively infected CD4+ T cells in secondary lymphatic tissues. However, if pyroptosis occurs in other peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients is currently unknown. We investigated if ASC speck positive cells are present in the circulation of HIV-1-infected patients. Design and methods: PBMCs or plasma of HIV-1 infected, antiretroviral therapy-naive patients were analyzed for the presence of ASC speck+ cells or extracellular ASC and compared with healthy controls. Intracellular staining for ASC was employed to detect ASC speck+ cells within PBMCs by flow cytometry, and ELISA to detect free ASC in the plasma. ASC multimerization was confirmed by immunoblot. Results: Peripheral blood CD14++CD16− monocytes were ASC speck+ in HIV patients, but not in healthy controls. In the subgroup analysis, HIV patients with lower CD4+ T-cell counts and higher viral load had significantly more ASC speck+ monocytes. ASC speck formation did not correlate with Gag expression, coinfection, lactate dehydrogenase or C-reactive protein. Conclusion: Our findings suggest that pyroptotic CD14++CD16− classical monocytes of HIV-1-infected patients release ASC specks into the blood stream, a phenomenon that may contribute to HIV-1 induced inflammation and immune activation.

LILRA3 deletion is a genetic risk factor of HIV infection

Objective: The aim of this study is to analyse the influence of LILRA3 and the genetic leukocyte immunoglobulin-like receptor 3 (LILRA3) deletion on transmission and clinical course of HIV infection. Design: Case and control study. Methods: LILRA3 genotypes were determined by PCR. HIV patients were categorized into short-term progressors, normal progressors and long-term nonprogressors according to the clinical course. Functional studies were performed using real-time PCR, intracellular flow cytometry and ELISA. Results: The prevalence of the homozygous LILRA3 deletion was higher in HIV-positive individuals (n = 439) than in controls (n = 651) (P = 0.02). The disease progression was faster in homozygously deleted patients with more short-term progressors than in heterozygous (P = 0.03) and homozygously positive (P = 0.002) individuals. These results have been confirmed in a seroconverter cohort (n = 288). The frequency of the homozygous deletion in the confirmation cohort was higher than in controls (P = 0.04). Combining both cohorts, the proportion of homozygously LILRA3-deleted individuals was 6.2% in HIV-infected patients (n = 727) vs. 3.2% in controls (P = 0.01). Functional analysis revealed an upregulation of the LILRA3 gene in real-time PCR in treated patients when compared with untreated patients (P = 0.007) and controls (P = 0.02) resulting in a higher LILRA3 expression in CD4+ (P = 0.008) and CD14+ (P = 0.02) cells of untreated patients in intracellular flow cytometry. LILRA 3 concentrations in the sera were similar between the groups, in untreated patients a correlation between viral load and LILRA3 concentration was found. Conclusion: The homozygous LILRA3 deletion is associated with a higher susceptibility for HIV disease and with a faster disease progression.

Brief Report: HIV-1 Infection Results in Increased Frequency of Active and Inflammatory SlanDCs that Produce High Level of IL-1β.

Abstract:HIV infection is marked by phenotypic and functional alterations of immune cells. Different studies have shown both numerical and functional deterioration of dendritic cells in HIV-1–infected patients. In this study, we report an increase of inflammatory 6-sulfo LacNAc dendritic cells (slanDCs) that are more activated and produce higher amounts of interleukin (IL)-1&bgr; during HIV-1 infection as compared with healthy controls. IL-1&bgr; plays a regulatory role in chronic inflammatory disorders. Therefore, our findings might reveal a compensatory regulatory function of slanDCs during HIV-1 infection.