Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Relationship between LAIR1 expression and intraleukocytic haemozoin. (a) Pairwise comparisons of LAIR1 transcript levels between children with and without pigment-containing monocytes (PCM) and pigment-containing neutrophils (PCN) in the non-SMA and SMA groups were determined using Mann-Whitney U test. Data are presented as box-plots, where the box represents the interquartile range, the line through the box is the median, and whiskers show the 10th and 90th percentiles. LAIR1 transcript levels were lower in children with PCM compared to those without, while LAIR1 transcript levels were comparable between absence/presence of PCN. (b) Temporal kinetics of LAIR1 transcript levels in response to Pf Hz treatment of PBMC from malaria-naïve donors ( n = 6, measured in triplicate). Pairwise comparisons were determined using Student t -test. Significant ( P < .05) differences in transcript levels between no treatment and Pf Hz treatment (10 μg/mL) groups represented by *at 0.5, 2, and 4 h time points. Data represent average of individuals ( n = 3) with each condition performed in triplicate (error bars represent SEM). (c) Immunoblot analysis of non-treated (baseline) and Pf Hz-treated (10 μg/mL) PBMC lysates for 12, 24, and 48 h. (d) Densitometric analysis of normalised cellular LAIR1 protein production presented as mean ± SEM. Across group comparisons analysed using ANOVA. Pairwise comparisons analysed using Student t-test. Data represent average of individuals ( n = 6) with each condition performed in triplicate (error bars represent SEM). LAIR1 protein levels were lower in Pf Hz-treated PBMC lysates compared to no treatment at 12, 24, and 24 h.

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques: Expressing, MANN-WHITNEY, Western Blot

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of intraleukocytic Pf Hz on LAIR1 and SHP-1 phosphorylation. Temporal kinetics of the signalling pathway in response to the different treatment conditions was determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). (a) Human Phospho-immunoreceptor array results showing LAIR1 and SHP-1 phosphorylation upon no treatment (baseline), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. Phosphorylation (spots) in the different conditions with control represented by blue boxes, LAIR1 by red boxes, and SHP-1 by green boxes. (b) Densitometric analysis of human phosphor-immunoreceptor array data. Data presented as (mean ± SEM). Across group comparisons analysed using ANOVA. *indicates significant differences ( P < .05) determined by Student t-test in pLAIR1 and pSHP-1 compared to C1q treatment. Phagocytosis of Pf Hz resulted in a reduction of pLAIR1 relative to C1q (alone) treatment. Similarly, ingestion of Pf Hz also caused a marked decrease in pSHP-1 levels relative to C1q treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques:

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: LAIR1 signalling pathway in malaria. The LAIR1 pathway is activated by attachment of collagen and collagenous ligands (C1q) to LAIR1 extracellular surface. This results in phosphorylation of intracellular LAIR1 ITIM tyrosine residues by Src family kinases. Phosphorylated ITIMs serve as docking sites for recruitment of SHP-1 and SHP-2 phosphatases. SHP-1 and SHP-2 become localized to phosphorylated ITIMs through their regulatory SH2 domains, subsequently inducing their phosphatase activity. Activated SHP-1 has been shown to block activation and nuclear translocation of nuclear factor nuclear factor-kappa beta (NF-κB) through de-phosphorylation of inhibitor of kappa-beta kinase complex (IKK). SHP-1 phosphatase activity also inhibits activation and translocation of IRFs from the cytoplasm to the nucleus by preventing TANK-binding kinase 1 (TANK-1) phosphorylation of interferon regulatory factors (IRFs). These events subsequently block transcription of inflammatory mediator encoding genes with response elements for IRFs and NF-κB. SHP-2 inhibits activation of IRF 8 and blocks expression of phagocyte NADPH oxidase (gp91 PHOX ). LAIR1 inhibitory signalling is regulated by soluble LAIR1 and LAIR2 (soluble homolog of LAIR1) through competition for collagenous ligands. Children with SMA had increased circulating levels of sLAIR1 and sLAIR2 indicative of enhanced receptor shedding. C1q was reduced in children with SMA, thereby, limiting ligand availability. Phagocytosis of Pf Hz antagonizes LAIR1 signalling through down-regulation of LAIR1 ITIM and SHP-1 phosphorylation, but does not alter SHP-2 phosphorylation. Leukocytic ingestion of Pf Hz also decreases LAIR1 transcripts and protein. These events result in NF-κB activation and the consequent production of pro-inflammatory mediators that enhance the pathogenesis of SMA. Red arrows represent ligand-receptor interaction, black solid arrows represent activation, and dashed black arrows represent blockade. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Soluble LAIR1 was measured in serum of study participants using the human LAIR1 ELISA matched antibody pair, and recombinant human LAIR1 (Creative Diagnostics, 45–16 Ramsey Rd, Shirley, NY 11967; Cat No. ABPR-0519).

Techniques: Activity Assay, Blocking Assay, Activation Assay, Translocation Assay, De-Phosphorylation Assay, Binding Assay, Expressing

Journal: medRxiv

Article Title: Polymerized type I collagen down-regulates STAT-1 phosphorylation through engagement to LAIR-1 in M1-macrophages avoiding long COVID

doi: 10.1101/2023.07.01.23292108

Figure Lengend Snippet: Binding of PTIC to LAIR-1 and its effect on M1-macrophages. (A) Binding assay of PTIC and native porcine type I collagen (CI) to LAIR-1. M1-macrophages treated with (B) anti-LAIR-1 antibody (1:100) for 24 hs or (D) anti-LAIR-1 antibody (1:100) and 10% PTIC for 24 h. Expression of CD14, CD16, CD163, IL-10 in cultures of M1-macrophage treated with (C) anti-LAIR-1 antibody or (E) anti-LAIR-1 antibody and 10% PTIC for 24 h. (F) M1 (CD16 + /CD36 + /CD86 + /IL-1β + ) and M2 (CD14 + /CD16 hi /CD163 + /IL-10 + ) cell percentage.

Article Snippet: Binding assays were performed by incubating various concentrations of recombinant human LAIR-1 (R&D #2664-LR-050) overnight at 4°C in 96 micro-wells plates coated with 5 μg/ml native porcine type I collagen or PTIC and blocked with 5% fat-free milk-PBS.

Techniques: Binding Assay, Expressing

Journal: medRxiv

Article Title: Polymerized type I collagen down-regulates STAT-1 phosphorylation through engagement to LAIR-1 in M1-macrophages avoiding long COVID

doi: 10.1101/2023.07.01.23292108

Figure Lengend Snippet: Binding of PTIC to LAIR-1 induces downregulation of STAT-1 phosphorylation. (A) Western blotting relative expression of STAT-1 and p-STAT1. (B) Relative expression of STAT-1 and pSTAT-1. Both were normalized for actin and were adjusted to 1 unit. (C) Effect of STAT-1 phosphorylation by activating LAIR-1 with anti-LAIR-1 antibody in M1-macrophages. (D) Relative expression of phosphorylated STAT-1. Results were normalized for actin and were adjusted to 1 unit.

Article Snippet: Binding assays were performed by incubating various concentrations of recombinant human LAIR-1 (R&D #2664-LR-050) overnight at 4°C in 96 micro-wells plates coated with 5 μg/ml native porcine type I collagen or PTIC and blocked with 5% fat-free milk-PBS.

Techniques: Binding Assay, Western Blot, Expressing