Fig 1.
hResistin/RELMα regulates priming and activation of the NLRP3 inflammasome.
hResistin/RELMα activates DAMP protein HMGB1, which activates NF-κB in macrophages, leading to the priming of NLRP3 and its associated proteins. hResistin/RELMα binds and activates BTK, allowing it to activate the NLRP3 inflammasome through phosphorylation of four critical tyrosines. BTK, Bruton’s tyrosine kinase; DAMP, damage-associated molecular pattern; HMGB1, high mobility group box 1; IL, interleukin; NLRP3, nucleotide-binding domain–like receptor protein 3; PH, pulmonary hypertension; PV-SMC, pulmonary vascular smooth muscle cell; RELMα, resistin-like molecule alpha. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0.
Table 1.
Hemodynamic data and diagnostic markers of patients with pulmonary hypertension.
Fig 2.
hResistin upregulates the expression of BTK, HMGB1, NLRP3, pro-IL-1β, pro-IL-18, and pro-caspase-1 and the cleavage of pro-IL-1β, pro-IL-18, and pro-caspase-1 in human macrophages.
Human THP-1–derived macrophages were cultured under the conditions shown for 24 hours. (A) Schematic diagram of proposed pathway. (B) Western blot images of HMGB1, NLRP3, BTK, pro- and cleaved caspase-1, pro- and cleaved IL-1β, and pro- and cleaved IL-18. (C) Quantitative analysis of each protein from panel B. Data represent means ± SD (n = 10 per group except the NLRP3 KO group, where n = 6). *p < 0.05, **p < 0.01, ****p < 0.0001. ab, antibody; Box-A, HMGB1 inhibitor; BTK, Bruton’s tyrosine kinase; Con, control; Ib, ibrutinib; HMGB1, high mobility group box 1; Hres, human resistin; IL, interleukin; KO, knockout; NLRP3, nucleotide-binding domain–like receptor protein 3; RAGE, receptor for advanced glycation end products. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0.
Fig 3.
hResistin stimulates NLRP3 inflammasome activation in human macrophages.
(A) Schematic presentation of proposed pathway. (B) Human macrophages were cultured in 6-well plates under the conditions shown for 15 minutes before being washed and lysed for western blot analysis with antibodies to phospho-tyrosine (p-Y) and NLRP3 (image is representative of 6 repeats). (C) Human macrophages were cultured under the conditions shown for 12 hours. PBS was used as a control. Cells were washed, lysed, and assayed for their ability to cleave a fluorescent caspase-1 substrate, YVAD-AFC. Values were normalized to PBS controls. All conditions were run in duplicate wells, and two independent experiments were performed. Error bars represent the mean ± SD (n = 4). ****p < 0.001 versus PBS. (D) Hypoxia upregulates BTK and NLRP3 colocalization in C57BL/6 WT mice but not in RELMα KO mice. Immunofluorescence images of NLRP3 and BTK in lung tissues of mice kept under normoxic or hypoxic conditions for 4 days. Lung sections were stained with anti-NLRP3 (red) and BTK (green). The arrowheads point to cells positively stained for BTK and NLRP3 (yellow). The upper images are shown at higher magnification (400×); the lower panels display separate channels. Representative photograph of n = 6 mice per group. (E) Human resistin colocalized with BTK and NLRP3 in patients with PH. Immunofluorescence images of lung tissue slices from PH patients. Sections were stained with anti-hResistin (red) and co-stained with anti-BTK (green) and anti-NLRP3 (cyan) antibodies. The arrowheads point to cells positively stained for hResistin, BTK, and NLRP3. Separate channels are displayed in the lower panels. Original magnification: 100 × , 200 × , and 400 × . 4DHx, 4-day hypoxic; ab, antibody; BTK, Bruton’s tyrosine kinase; Con, control; Hres, human resistin; Ib, ibrutinib; KO, knockout; NLRP3, nucleotide-binding domain–like receptor protein 3; Nx, normoxic; PBS, phosphate-buffered saline; PH, pulmonary hypertension; RELMα, resistin-like molecule alpha; WT, wild-type. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0.
Fig 4.
RELMα upregulates proinflammatory phenotypes in hypoxic mouse lungs.
(A) Quantitative RT-PCR analysis of HMGB1, BTK, NLRP3, pro-IL-1β, and RELMα gene expression in lung tissues collected from WT C57BL/6 mice that were kept for 4 days under hypoxic conditions with or without hResistin antibody or control antibody. Data represent means ± SD (n = 6 per group). (B) Quantitative RT-PCR analysis of HMGB1, BTK, and NLRP3 gene expression in WT and RELMα KO mouse lungs after exposure to normoxic and hypoxic conditions (n = 6 per group). (C) Western blot images show protein expression of BTK, HMGB1, and NLRP3 in lungs from WT and RELMα KO mice after exposure to normoxic and hypoxic conditions. (D) Quantitative analysis of data in panel C. Data represent means ± SD (n = 6 per group). ***p < 0.001, ****p < 0.0001. 4DHx, 4-day hypoxic; BTK, Bruton’s tyrosine kinase; FC, fold change; HMGB1, high mobility group box 1; hResistin, human resistin; KO, knockout; NLRP3, nucleotide-binding domain–like receptor protein 3; Nx, normoxic; RELM, resistin-like molecule; WB, western blot; WT, wild-type. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0.
Fig 5.
Macrophages are the main source of NLRP3 in hypoxic mouse lungs and PH patient lungs.
(A) Immunofluorescence images of NLRP3 and immune cell markers in lung tissues of 4-day hypoxic mice and lung sections from PH patients. Lung sections were stained with anti-NLRP3 (red) and/or Mac2 (green), MPO (green), or CD79b (green). The images are shown at 100 × magnification. Representative photograph of 6 mice per group and 3 human lung samples. (B) Immunofluorescence images of lung tissue slices from PH patients show hResistin colocalization with NLRP3 in macrophages. Sections were stained with anti-human resistin (red) and co-stained with anti-Mac2 (green) and anti-NLRP3 (cyan) antibodies. The arrowheads point to cells positively stained for hResistin, Mac2, and NLRP3. Separate channels are displayed in the lower panels. Original magnification: 100 × , 200 × , and 400 × . (C) Immunofluorescence images show colocalization of NLRP3 and BTK in macrophages. Sections were stained with anti-human BTK (green) and co-stained with anti-Mac2 (red) and anti-NLRP3 (cyan) antibodies. The arrowheads point to cells positively stained for human macrophages, BTK, and NLRP3. Separate channels are displayed in the lower panels. Original magnification: 400 × . BTK, Bruton’s tyrosine kinase; hResistin, human resistin; MPO, myeloperoxidase; NLRP3, nucleotide-binding domain–like receptor protein 3; PH, pulmonary hypertension. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0.
Fig 6.
hResistin induces macrophage-derived mature IL-1β and IL-18 to promote human PVSMC proliferation.
(A) Images of western blots for p-AKT, t-AKT, p-ERK1/2, and t-ERK1/2 protein levels in human PVSMCs that were serum-starved for 24 hours and then cultured for 30 minutes in (1) medium only; conditioned medium from macrophages treated with (2) 20nM hResistin, (3) 20 nM hResistin + 300 ng/mL resistin antibody, (4) 20 nM hResistin + 10 µM (4.04 µg/mL) MCC950, (5) 120 ng/mL IL-1β antibody, (6) or 120 ng/mL IL-18 antibody; (7) starved media (0.5% FBS) + 5 ng/mL IL-1β protein; or (8) starved media + 5 ng/mL IL-18 protein. (B) Quantitative analysis of data in panel A. Data represent means ± SD (n = 5 per group). (C) Induction of PVSMC proliferation by IL-1β and IL-18 derived from hResistin-treated macrophages. Data represent means ± SD (n = 6 per group). (D) hResistin regulates MMP-driven PVSMC proliferation through IL-1β and IL-18 derived from hResistin-treated macrophages. Human PVSMCs were serum-starved for 24 hours and then cultured for 24 hours with (1) medium only; conditioned media from macrophages treated with (2) 20 nM hResistin, (3) 120 ng/mL IL-1β antibody, or (4) 120 ng/mL IL-18 antibody; (5) starved media + 5 ng/mL IL-1β protein; (6) starved media + 5 ng/mL IL-18 protein; or (7) conditioned media alone. (E) Quantitative analysis of data in panel D. Data represent means ± SD (n = 4 per group). (F) Schematic presentation of the proposed pathway of SMC migration and proliferation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ab, antibody; CM, conditioned medium; con, control; FBS, fetal bovine serum; Hres; human resistin; IL, interleukin; NS-CM, non-stimulated conditioned media; MMP, matrix metalloproteinase; OD, optical density; p-, phosphorylated; PDGF, platelet-derived growth factor; PVSMC, pulmonary vascular smooth muscle cell; t-, total. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0.