CD163, a novel receptor for TNF, was revealed in situ by proximity ligation assay

Abstract

Cytokine therapy utilizes cytokines to enhance the immune system to fight diseases. These strategies rely on advanced knowledge, including the communication between cytokines and their receptors. In situ, cytokine-receptor interactions are typically analyzed by co-localization using immunolabeling. Our study compared co-localization using the Proximity Ligation Assay (PLA), a recently developed in situ protein-protein interaction technique. In an inflamed porcine lung model, we demonstrated the efficacy of PLA in detecting interactions between tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2. Additionally, the CD163 receptor was identified as a novel partner of TNF. Furthermore, the combination of immunolabeling and PLA offered additional insights, particularly, the internalization of TNF following its binding with CD163 in macrophages. Our work focused on in situ interactions of TNF with macrophages TNF receptors and suggested exciting perspectives for further understanding and application of cytokine-based therapies.

Keywords: CD163; Inflammation; Proximity ligation assay; Regeneration; SPR; TNF.

Graphical abstract

Fig. 1

Macroscopic morphology of inflamed lung tissue of PIG-4. (A-E) HPS-stained lung sections. (B-E) Magnified view: higher magnification images of lung morphology from four representative areas within the black rectangle of image A. (B) Zone 1, tissue around the necrosis; (C) Zone 2, inflamed bronchioles; (D) Zone 3, inflamed tissue where the native histological lung structure was not detectable; and (E) Zone 4, tissue still containing alveoli lung structure.

Fig. 2

Co-localization of TNF and four receptors in the inflamed lung zones in situ. (A,E,I,M) Immunolabeling of both TNF and TNFR1; (B,F,J,N) both TNF and TNFR2; (C,G,K,O) both TNF and CD163 and (D,H,L,P) both TNF and CD206. (A-D) Zone 1, (E-H) Zone 2; (I-L) Zone 3, and (M-P) Zone 4. Quantitative analysis of the expression of (Q) TNF and TNFR1, (R) TNF and TNFR2, (S) TNF and CD163, (T) TNF and CD206 in the different regions. Quantitative analysis based on random examination of 3 sets of 1000 cells per condition. Percentage of colocalization. Anti-TNFR1, anti-TNFR2, anti-CD163, anti-CD206 (Alexa Fluor 488, cyan), anti-TNF (Alexa Fluor 568, red fluorescence). Nuclear staining with DAPI (blue fluorescence). Autofluorescence of the blood red cells (magenta). Scale bar = 50 μm. Arrow: co-localization of the immunolabeling.

Fig. 3

Interaction between different cytokines and receptors in situ. (A,E,I,M) Proximity Ligation Assay with TNF and TNFR1; (B,F,J,N) with TNF and TNFR2; (C,G,K,O) with TNF and CD163 and (D,H,L,P) with TNF and CD206. (A-D) Zone 1, (E-H) Zone 2; (I-L) Zone 3, and (M-P) Zone 4. (Q) Quantitative analysis of the interaction between cytokine and receptors in the different regions. Quantitative analysis based on random examination of 3 sets of 1000 cells per condition. Percentage of interactions, in red significant difference between Co-localization and IPP quantification (p < 0.05, t-test). Cytokine/receptor interaction in yellow. Nuclear staining with DAPI (blue fluorescence). Autofluorescence of the blood red cells (magenta). Scale bar = 50 μm. Arrow: interaction between cytokine and receptor.

Fig. 4

Combination of immunolabeling with IPP. (A) IPP between TNF and CD163 proteins. (B) IPP between TNF/CD163 and immunolabeling with anti-Iba1 antibody. (C) IPP between TNF/CD206 and immunolabeling with anti-Iba1 antibody. (D) Negative control for IPP between TNF/CD163, IPP between TNF/CD206, and immunolabeling with anti-Iba1 antibody. Cytokine/receptor interaction in yellow. Anti-Iba1 (Alexa Fluor 488, cyan). Nuclear staining with DAPI (blue fluorescence). Scale bar = 10 μm. Arrow flat: PPI of the TNF/CD163. Arrow: Iba1 immunolabeling.

Fig. 5

Our findings indicate that CD163 may serve as a decoy receptor for TNF, potentially modulating inflammation by binding to TNF or related ligands without initiating downstream signaling. By acting as a decoy, CD163 could block TNF from engaging with its functional receptors, thereby dampening inflammatory responses.