Journal: Frontiers in Immunology

Article Title: Attenuated Negative Feedback in Monocyte-Derived Macrophages From Persons Living With HIV: A Role for IKAROS

doi: 10.3389/fimmu.2021.785905

Figure Lengend Snippet: Impaired expression of RELA and IKZF1 in PLWH-MDMs compared to controls. (A) Relative expression of RELA mRNA calculated over T0. Error bars indicate SEM; *indicates statistical significance between groups (Ctrls, n=10; PLWH, n=25). (B) Expression of RELA protein in MDMs obtained from Ctrls (n=4) and PLWH (n=9) treated with GM-CSF (G) and LPS (L). Blots were quantified using image J software. The graph represents the ratio of RELA in LPS compared to GM-CSF after normalization with GAPDH (right panel). *Indicates statistical significance between Ctrls and PLWH. (C) Relative expression of IKZF1 in controls (n=10) and PLWH (n=25). The tables report specific P -values calculated within each group and between groups.

Article Snippet: PLWH-derived MDM cells cultured for 5 days were transfected with 3 μg IKZF1 plasmid (pLV[Exp]-EGFP:T2A:Puro-CMV>hIKZF1[NM_006060.5, VectorBuilder, Chicago, IL) or the empty vector BFP (pLenti6.2-mTagBFP2, Addgene #113725) using the ViaFect transfection reagent (Promega; Madison, WI).

Techniques: Expressing, Software

Journal: Frontiers in Immunology

Article Title: Attenuated Negative Feedback in Monocyte-Derived Macrophages From Persons Living With HIV: A Role for IKAROS

doi: 10.3389/fimmu.2021.785905

Figure Lengend Snippet: Transfection of IKZF1 into PLWH-MDMs reduces their hyper-responsiveness to LPS and partially restores the expression of negative regulators of inflammation. (A) Relative expression of IKZF1 mRNA in IKZF1-transfected PLWH-MDMs (left panel, n=4) at day 3 or day 5 post-isolation (right panel, n=4) in the indicated treatments calculated over T0. BFP control vector or untransfected cells were used as negative controls at day 3 or day 5, respectively. Expression levels of pro-inflammatory cytokines TNFA , IL6 , and IL1B (B) and negative regulators of inflammation NCOR2 , GSN , HIVEP1 , BIN1 , and IDO1 (C) in cells transfected at day 5 post-isolation (See Figure S1 for data obtained after transfection at day 3). In all graphs, error bars indicate SEM; *indicates statistical significance between groups.

Article Snippet: PLWH-derived MDM cells cultured for 5 days were transfected with 3 μg IKZF1 plasmid (pLV[Exp]-EGFP:T2A:Puro-CMV>hIKZF1[NM_006060.5, VectorBuilder, Chicago, IL) or the empty vector BFP (pLenti6.2-mTagBFP2, Addgene #113725) using the ViaFect transfection reagent (Promega; Madison, WI).

Techniques: Transfection, Expressing, Isolation, Control, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Attenuated Negative Feedback in Monocyte-Derived Macrophages From Persons Living With HIV: A Role for IKAROS

doi: 10.3389/fimmu.2021.785905

Figure Lengend Snippet: Schematic illustration of an imbalanced immune response in PLWH. In healthy controls, temporally-regulated positive (miR-155-5p, IDO1, IL6, TNFA and IL1B) and negative feedbacks (146a-5p, NCOR2, GSN, MSC, IKZF1, BIN1, ISG20, and RELA) will lead to a balanced immune response, with IKZF1 and RELA playing a central role in the transcription of negative regulators. Instead, attenuated IKAROS/RELA axis in PLWH-MDMs results in positive signals dominating over negative feedback. This imbalanced immune response may contribute to chronic inflammation and increase the risk of co-morbidities.

Article Snippet: PLWH-derived MDM cells cultured for 5 days were transfected with 3 μg IKZF1 plasmid (pLV[Exp]-EGFP:T2A:Puro-CMV>hIKZF1[NM_006060.5, VectorBuilder, Chicago, IL) or the empty vector BFP (pLenti6.2-mTagBFP2, Addgene #113725) using the ViaFect transfection reagent (Promega; Madison, WI).

Techniques: