Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: Decomposition of the inflammatory score was conducted by estimating the most variable jacobians (first order partial derivative of inflammatory age) ( A ). Both positive and negative contributors to inflammatory age are observed. The top 15 most variable jacobians are CXCL9, EOTAXIN, Mip-1α, LEPTIN, IL-1β, IL-5, IFN-α and IL-4 (positive contributors) and TRAIL, IFN-β, CXCL1, IL-2, TGF-α, PAI-1 and LIF (negative contributors) (A). In a validation study, 97 healthy adults (age 25-90) well matched for cardiovascular risk factors, were selected form a total of 151 recruited subjects. Immune protein analysis was conducted in samples from these subjects. CXCL9, HGF, CXCL1 and LIF were found to change in the same direction in both the Stanford 1KIP and the validation cohort ( A and B ). Cardiovascular age was estimated using 3 parameters: (1) aortic pulse wave velocity, a measure of vascular stiffness; (2) relative wall thickness (RWT), a measure of ventricular remodeling, and (3) early diastolic mitral annular velocities (e’), a measure of ventricular relaxation. After adjusting for age, sex, BMI, heart rate, systolic blood pressure, fasting glucose and total cholesterol to HDL ratio, positive correlations were obtained between CXCL9 and PWV ( R = 0.22) and RWT ( R = 0.3) ( P < 0.05), and negative correlations were observed between LIF and PWV ( R = −0.27), and RWT ( R = −0.22) ( C and D ). No variable included in the models had high co-linearity as suggested by variance inflation factors (VIF) < 3 for each factor. Induced pluripotent stem cells (hiPSCs) were obtained from isolated fibroblasts ( N = 5, in duplicates) using the Yamanaka factors and differentiated them into endothelial cells (hiPSC-ECs) under well-defined conditions as previously described . Expression levels of CXCL9 and SIRT3 were measured by RT-PCR as described under Methods. A significant age-dependent increase in CXCL9 mRNA expression levels is observed ( P < 0.01), which reaches a plateau after the sixth cell passage ( E ). Concomitant with the increase in CXCL9, down-regulation in SIRT3 mRNA can be observed after the second cell passage ( P < 0.01) (E). Addition of increasing doses (10 to 800 ng/ml) of exogenous CXCL9 to young (day 7) hiPSC-ECs induces down-regulation of SIRT3 mRNA expression (F). Expression of the CXCL9 receptor, CXCR3, was measured in young cardiomyocytes derived from hiPSCs (hiPSC-CM) as well as in hiPSC-ECs, HUVEC cells, freshly isolated fibroblasts and hiPSCs. Elevated expression is observed in hiPSC-ECs, HUVEC cells but not in other cell types ( F ) suggesting that the endothelium but no other cell subsets is target of CXCL9 and potentially other CXCR3 ligands as well.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: Biomarker Discovery, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: To validate the association between CXCL9 expression and SIRT3 in hiPSC-ECs, CXCL9 was knockdown (KD) in hiPSCs using shRNA. Quantitative PCR data show significant knockdown of CXCL9 in hiPSCs following transfection with CXCL9-shRNA compared to scramble controls ( A ). hiPSC-ECs failed to show an increase in CXCL9 expression in CXCL9-KD cells as they were passaged when compared to scramble controls ( B ). On the contrary, CXCL9-KD hiPSC-ECs show reversal of their SIRT3 levels in serially passaged hiPSC-ECs when compared to scramble controls ( C ). Quantification of the number of capillary-like networks formed by scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubular networks even at later passages when compared to scramble controls that showed impaired tube formation at later passages of hiPSC-ECs ( D ). Line graph of percent relaxation of mouse thoracic aortic sections to Acetylcholine show impaired vascular reactivity to increasing concentrations of CXCL9, suggesting CXCL9 impairs vascular function ( E ). Significance of impaired vascular reactivity was determined by 2-way ANOVA, followed by a Bonferroni post-test, N=3, n=3-4, *p<0.05.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: Expressing, Knockdown, shRNA, Real-time Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: Representative images of capillary-like networks from scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubes even at later passages when compared to scramble that showed impaired tube formation towards later passages of hiPSC ECs.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques:

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation

doi: 10.1152/ajplung.00056.2018

Figure Lengend Snippet: Real-time PCR mouse primers used to assess gene expression in this study

Article Snippet: A solution containing 10 5 PMN in 100 μl was placed in the upper chamber, and PMN were stimulated with keratinocyte chemoattractant ( KC ; 50 ng/ml, Peprotech), N -formyl-methionyl-leucyl-phenylalanine (fMLP; 10 5 M, Sigma-Aldrich), and recombinant mouse Cxcl9 (50 ng/ml, R&D Systems) and Cxcl10 (100 ng/ml, BioLegend).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation

doi: 10.1152/ajplung.00056.2018

Figure Lengend Snippet: Immune response and host defense gene expression differences in Bpifa1 −/− mice during acute inflammation

Article Snippet: A solution containing 10 5 PMN in 100 μl was placed in the upper chamber, and PMN were stimulated with keratinocyte chemoattractant ( KC ; 50 ng/ml, Peprotech), N -formyl-methionyl-leucyl-phenylalanine (fMLP; 10 5 M, Sigma-Aldrich), and recombinant mouse Cxcl9 (50 ng/ml, R&D Systems) and Cxcl10 (100 ng/ml, BioLegend).

Techniques: Gene Expression, Migration

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation

doi: 10.1152/ajplung.00056.2018

Figure Lengend Snippet: BPI fold-containing group A member 1-deficient (Bpifa1−/−) polymorphonuclear neutrophils (PMN) do not have impaired chemotaxis response. PMN from wild-type (WT) and Bpifa1−/− mice were isolated from bone marrrow. Chemotaxis assays were performed using a modified Boyden technique. WT and Bpifa1−/− PMN were stimulated with PMN chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP; 105 M), keratinocyte chemoattractant (KC; 50 ng/ml), C-X-C motif chemokine ligand 10 (Cxcl10; 100 ng/ml), Cxcl9 (50 ng/ml), and combination of Cxcl10 and Cxcl9. Total PMN migration represented as relative fluorescence units (RFU). ●, WT PMN; ○, Bpifa1−/− PMN; n = 4–6 wells/group. Generalized linear mixed model: no significant differences observed. Horizontal bars indicate mean values. Data represent 2 experiments.

Article Snippet: A solution containing 10 5 PMN in 100 μl was placed in the upper chamber, and PMN were stimulated with keratinocyte chemoattractant ( KC ; 50 ng/ml, Peprotech), N -formyl-methionyl-leucyl-phenylalanine (fMLP; 10 5 M, Sigma-Aldrich), and recombinant mouse Cxcl9 (50 ng/ml, R&D Systems) and Cxcl10 (100 ng/ml, BioLegend).

Techniques: Chemotaxis Assay, Isolation, Modification, Migration, Fluorescence

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BPIFA1 regulates lung neutrophil recruitment and interferon signaling during acute inflammation

doi: 10.1152/ajplung.00056.2018

Figure Lengend Snippet: Diagram: BPI fold-containing group A member 1 (Bpifa1) regulation of noncanonical Toll-like receptor 4 (TLR4) and interferon (IFN) signaling. Left: Bpifa1 is a lipopolysaccharide (LPS)-binding molecule and is likely to influence inflammatory signaling by modulating LPS-TLR4 interactions. In the absence of Bpifa1, impaired noncanonical TLR4 signaling through TIR domain-containing adapter-inducing INFβ1 (TRIF) results in decreased interferon regulatory factor 7 (IRF7) expression, leading to decreased IFNλ expression. Canonical LPS-TLR4 activation and cytokine expression is not affected by Bpifa1 deficiency. Middle: Bpifa1 binding to LPS facilitates immune signaling through TNF receptor associated factors (TRAF), which in turn activates IRF5 to induce interleukin (IL)-12 expression, inducing IFNγ. During Bpifa1 deficiency, key components of this pathway are downregulated (IL-12rb1, Jak2, Irf5), resulting in decreased IFNγ expression. Right: decreased expression of IFNγ and IFNλ during Bpifa1 deficiency causes downstream downregulation of Irf9, Irf7, Cxcl10, Cxcl9 and IFN-stimulated genes (ISG). Shaded boxes denote lower gene expression in our model. Dashed arrows represent impaired signaling pathways in our model. AEC, airway epithelial cell; APC, antigen-presenting cell; AL, airway lumen; NF-κB, nuclear factor-κB; TNFα, tumor necrosis factor-α; JAK, Janus kinase; STAT, signal transducer and activator of transcription; CXCL, C-X-C motif chemokine ligand.

Article Snippet: A solution containing 10 5 PMN in 100 μl was placed in the upper chamber, and PMN were stimulated with keratinocyte chemoattractant ( KC ; 50 ng/ml, Peprotech), N -formyl-methionyl-leucyl-phenylalanine (fMLP; 10 5 M, Sigma-Aldrich), and recombinant mouse Cxcl9 (50 ng/ml, R&D Systems) and Cxcl10 (100 ng/ml, BioLegend).

Techniques: Binding Assay, Expressing, Activation Assay, Gene Expression, Protein-Protein interactions