normal human lung fibroblasts mrc 5  (ATCC)

Journal: Frontiers in Immunology

Article Title: CD14 + CD16 + monocyte-derived TREM2 macrophages promote lung fibrosis in systemic sclerosis–interstitial lung disease

doi: 10.3389/fimmu.2026.1748574

Figure Lengend Snippet: GM-CSF-induced CD14 + CD16 + monocyte-derived macrophages promoted fibrotic activation of lung fibroblasts, an effect depends upon TREM2 expression. (A) Volcano plot showing DEG in fibroblasts from SScLO compared to those from HCs ( GSE128169 ). Fibrotic activation genes, including POSTN , COL1A1 , COL3A1 , COL4A1 , COL5A1 , COL8A1 , TGFBI , and ACTA2 , were significantly upregulated in SScLO fibroblasts. (B) Schematic diagram of the co-culture experimental design. Monocytes (CD14 + CD16 - or CD14 + CD16 + ) were differentiated into macrophages using GM-CSF for 7 days, then co-cultured with human lung fibroblasts for 5 days with or without TREM2 inhibitor. Bulk RNA sequencing and western blot analyses were performed to assess gene expression and protein levels, respectively. (C) Volcano plots showing DEG in fibroblasts co-cultured with CD14 + CD16 - vs. CD14 + CD16 + macrophages (left) and CD14 + CD16 + macrophages with or without TREM2 inhibitor (right). All macrophages were derived from SSc-ILD patients. Co-culture with CD14 + CD16 + macrophages induced fibrotic gene expression ( POSTN , COL7A1 , ACTA2 , and TGFBI ), which was significantly reduced by TREM2 inhibition. Upregulated genes are shown in red (FC > 2, adj. p < 0.05), downregulated in blue (FC < -2, adj. p < 0.05), and non-significant genes in black. Dotted lines indicate statistical thresholds. (D) Representative western blot images of COL7A1 (34 kDa), COL3A1 (200 kDa), COL1A1 (220 kDa), and α-SMA (42 kDa); GAPDH (37 kDa) was used as a loading control. Conditions with TREM2 inhibitor are indicated in red. (E–H) Quantification of protein levels normalized to GAPDH: (E) COL7A1, (F) COL3A1, (G) COL1A1, and (H) α-SMA. Expression of COL7A1, COL1A1, and α-SMA was significantly elevated in fibroblasts co-cultured with CD14 + CD16 + monocytes derived macrophages from SSc-ILD compared to HCs and CD14 + CD16 - monocytes derived macrophages from SSc-ILD. COL3A1 expression was also elevated in SSc-ILD fibroblasts compared to HCs. TREM2 inhibition markedly reduced expression of all fibrotic markers. Statistical analysis was conducted using MANOVA, followed by Tukey’s post-hoc multiple comparisons test. Data are presented as mean ± SEM, with n = 6 per group. Statistical significance is indicated as * p < 0.05, ** p < 0.01 for comparisons between SSc-ILD and HCs within the same subset; # p < 0.05, ## p < 0.01 for comparisons between CD14 + CD16 - and CD14 + CD16 + subsets within SSc-ILD patients; $ p < 0.05, $$ p < 0.01 for comparisons between SSc-ILD and TREM2 inhibitor within the same subset.

Article Snippet: Macrophages differentiated from monocyte subsets by GM-CSF from SSc-ILD patients were cultured for 7 days and subsequently co-cultured with commercially sourced human lung fibroblasts (ACBR 469, Cell Systems, Kirkland, WA, USA) for 5 days, with or without treatment with a TREM2 inhibitor (BLP-NR018, Alomone Labs, Jerusalem, Israel).

Techniques: Derivative Assay, Activation Assay, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Western Blot, Gene Expression, Inhibition, Control

Journal: Frontiers in Immunology

Article Title: CD14 + CD16 + monocyte-derived TREM2 macrophages promote lung fibrosis in systemic sclerosis–interstitial lung disease

doi: 10.3389/fimmu.2026.1748574

Figure Lengend Snippet: TREM2 macrophages and fibrotic progression in SSc-ILD. This schematic illustrates the proposed mechanism by which GM-CSF–induced monocyte-derived macrophages differentiate and promote lung fibrosis in patients with SSc-ILD. In SSc-ILD, recruited monocytes stimulated by GM-CSF upregulate TREM2 and GPNMB , leading to increased secretion of cytokines such as CCL2 and TGF-β1. CCL2 promotes additional recruitment of monocytes via the CCL2-CCR2 axis. These TREM2 + macrophages enhance fibroblast activation through TGF-β1 production, driving fibroblast to myofibroblast differentiation and contributing to fibrotic remodeling in the lung.

Article Snippet: Macrophages differentiated from monocyte subsets by GM-CSF from SSc-ILD patients were cultured for 7 days and subsequently co-cultured with commercially sourced human lung fibroblasts (ACBR 469, Cell Systems, Kirkland, WA, USA) for 5 days, with or without treatment with a TREM2 inhibitor (BLP-NR018, Alomone Labs, Jerusalem, Israel).

Techniques: Derivative Assay, Activation Assay

Journal: bioRxiv

Article Title: Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

doi: 10.64898/2026.06.01.729382

Figure Lengend Snippet: A) Hydrogels were composed of either 0% or 50% mixture of tyramine-modified silk and were exposed to media without or with 5 ng/mL TGFβ. After hydrogels were cast, 25,000 NHLF cells were seeded on the surface. B) SEM imaging of the surface of the silk hydrogels incubated without cells for Day 0 and Day 14. C) Live/Dead imaging of Calcein AM for live cells (green) and Ethidium Homodimer-1 for dead cells (red) indicated cytocompatibility. D) Alamar Blue Assay for metabolic activity indicated that 0% gels showed an increase in activity over 14 days, while 50% gels plateaued from day 7 to 14. E) Mammalian β-Galactosidase Assay for senescence revealed no change for any timepoints tested over 14 days. N=3-4, Error bars = SEM.TCP = Tissue Culture Plate, T-= without TGFβ, T+ = 5 ng/mL of TGFβ. Two-way ANOVA, Tukey’s: ns = not significant p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Normal human lung fibroblasts (NHLFs, PCS-201-013, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM, ThermoFisher) supplemented with 10% Gibco Fetal Bovine Serum, Premium (FBS, ThermoFisher), 1% Gibco non-essential amino acids (ThermoFisher), and 1% Gibco antibiotic–antimycotic (ThermoFisher).

Techniques: Modification, Imaging, Incubation, Alamar Blue Assay, Activity Assay

Journal: bioRxiv

Article Title: Progressive matrix stiffening of tyramine-modified silk fibroin hydrogels governs stage-specific pulmonary fibroblast activation

doi: 10.64898/2026.06.01.729382

Figure Lengend Snippet: Top row: NHLFs on 0% SF-TA hydrogels at Day 2, Day 7, and Day 14, exhibiting a proliferative, inflammatory phenotype consistent with early fibroblast activation, characterized by secretion of IL-6, IL-8, and MCP-1 (dots) and progressive collagen deposition (fibers). Bottom row: NHLFs on 50% SF-TA hydrogels at Day 2, Day 7, and Day 14, exhibiting metabolic plateau and lower inflammatory cytokine secretion, gradual collagen secretion, with TGFβ-dependent myofibroblast activation. Together, these two substrate conditions recapitulate distinct stages of the fibrotic disease continuum.

Article Snippet: Normal human lung fibroblasts (NHLFs, PCS-201-013, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium, high glucose (DMEM, ThermoFisher) supplemented with 10% Gibco Fetal Bovine Serum, Premium (FBS, ThermoFisher), 1% Gibco non-essential amino acids (ThermoFisher), and 1% Gibco antibiotic–antimycotic (ThermoFisher).

Techniques: Activation Assay