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. 2023 Nov 24:14:1250920.
doi: 10.3389/fimmu.2023.1250920. eCollection 2023.

Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis

Ruiqing Wang  1 , Xinyue Ma  1 , Xinyu Zhang  1 , Dizhi Jiang  1 , Hongyuan Mao  1 , Zerun Li  1 , Yu Tian  1 , Bo Cheng  1
Affiliations

Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis

Ruiqing Wang et al. Front Immunol. .

Abstract

Introduction: Radiation pneumonitis is a critical complication that constrains the use of radiation therapy for thoracic malignancies, leading to substantial morbidity via respiratory distress and lung function impairment. The role of Natural killer (NK) cells in inflammatory diseases is well-documented; however, their involvement in radiation pneumonitis is not fully understood.

Methods: To explore the involvement of NK cells in radiation pneumonitis, we analyzed tissue samples for NK cell presence and function. The study utilized immunofluorescence staining, western blotting, and immunoprecipitation to investigate CXCL10 and ROS levels, autophagy activity, and NKG2D receptor dynamics in NK cells derived from patients and animal models subjected to radiation.

Result: In this study, we observed an augmented infiltration of NK cells in tissues affected by radiation pneumonitis, although their function was markedly diminished. In animal models, enhancing NK cell activity appeared to decelerate the disease progression. Concomitant with the disease course, there was a notable upsurge in CXCL10 and ROS levels. CXCL10 was found to facilitate NK cell migration through CXCR3 receptor activation. Furthermore, evidence of excessive autophagy in patient NK cells was linked to ROS accumulation, as indicated by immunofluorescence and Western blot analyses. The association between the NKG2D receptor and its adaptor proteins (AP2 subunits AP2A1 and AP2M1), LC3, and lysosomes was intensified after radiation exposure, as demonstrated by immunoprecipitation. This interaction led to NKG2D receptor endocytosis and subsequent lysosomal degradation.

Conclusion: Our findings delineate a mechanism by which radiation-induced lung injury may suppress NK cell function through an autophagy-dependent pathway. The dysregulation observed suggests potential therapeutic targets; hence, modulating autophagy and enhancing NK cell activity could represent novel strategies for mitigating radiation pneumonitis.

Keywords: CXCL10/CXCR3; NK cell; NKG2D; autophagy; radiation pneumonitis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Radiation pneumonia is accompanied by an accumulation of NK cells and a decline in their functionality. Immunofluorescence staining (A) and flow cytometry analysis (B) for detecting the number of NK cells. Scale bar (left) = 50 μm, Scale bar (right) = 25 μm. (C) Flow cytometry analysis for the expression of functional indicators (IFN-γ and granzyme B) of lung tissue-infiltrating NK cells. (D, E) The apoptosis rate and CCK8 results indicated that the vitality of NK cells was significantly weaker when mice had a radiation pneumonia. (F, G) The levels of IFN-γ and granzyme B in the culture supernatant of NK cells. (A–C, F, G): n = 5; (D, E): n = 3. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. ***P = 0.0003 (A), **P = 0.001 (B), ***P < 0.0001 IFN-γ, ***P = 0.0008 Gran B (C), **P = 0.0023 (D), ***P = 0.0002 72h, **P = 0.0016 96h (E), ***P < 0.001 (F), ***P < 0.001 (G).
Figure 2
Figure 2
Lung injury promotes the recruitment of NK cells via the CXCL10/CXCR3 pathway. NK cell-associated chemokines in mouse lung tissue (A) and BALF (B) were detected respectively. PCR (C, E) as well as Western blotting analysis (D, F) revealed that NK cells from the lungs of RP group were markedly activated at both the RNA and protein levels. (G) Demonstration of AMG487 reversing radiotherapy-induced decline in NK cell count in mice. Scale bar (left) = 50 μm, Scale bar (right) = 50 μm. (H) Transwell assay depicting NK cell migration under control, radiotherapy, and radiotherapy with AMG487. Scale bar = 50 μm (A-G): n = 3. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. *P = 0.0142 CCL3, **P = 0.0033 CCL4, **P = 0.0090 CCL5, ***P = 0.0002 CXCL8, ***P = 0.0005 CXCL10, *P = 0.0358 CXCL12 (A), ***P < 0.0001 CCL5, ***P = 0.0001 CXCL10 (B), ***P = 0.0003 (C), *P = 0.0118 (D), **P = 0.0048 RhoA, ***P< 0.0001 MAPK (E), **P = 0.0044 RhoA, **P = 0.0038 MAPK (F).
Figure 3
Figure 3
Overactive Autophagy in Lung NK Cells Induced by Radiation. (A) Volcano Plot of Differentially Expressed Genes: This plot shows the relationship between gene expression levels and statistical significance, highlighting genes that are differentially expressed between two conditions. The x-axis represents the log-transformed expression levels of genes, the y-axis represents the negative logarithm of statistical significance, and the scatter plot represents differentially expressed genes. Red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots indicate genes with no significant difference. (B) Number of Differentially Expressed Genes: Comparison of the number of differentially expressed genes detected in RNA-seq analysis under different conditions. The numbers in the table represent the count of genes significantly upregulated or downregulated in each condition. (C) Pathway enrichment analysis of the differentially expressed gene set using the KEGG database. This plot shows the differentially expressed genes enriched KEGG pathways. (D) Compared to the control group, multiplex immunofluorescence staining showed that autophagy was excessively activated in the lung NK cells of the RP group mice. Scale bar = 20 μm. (E–G) RT-qPCR and Western blotting results indicated an increase in the expression of autophagy-related proteins in lung NK cells from the RP group mice. (D–G): n = 3. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. **P = 0.0014 (D), ***P < 0.001 LC3, **P = 0.0031 ATG5 (E), **P = 0.0018 LC3, ***P = 0.0002 ATG5 (G).
Figure 4
Figure 4
Restoration of NK Cell Function through Inhibition of ROS Accumulation or Excessive Autophagy. (A) Using mitochondrial ROS-specific fluorescent dye (MitoSOX red) to observe the distribution of mitochondrial ROS. (B) The effect of NAC on the expression of autophagy-related protein (LC3) in NK cells as demonstrated by Western blotting. (C) HE-staining of lung tissue from mice exposed to 20Gy irradiation with or without autophagy inhibitor treatment. Left: 20x magnification. Right: 40x magnification. ROS inhibition reduces radiation-induced lung damage. (D) As demonstrated by CCK8 assays, radiation-induced excessive autophagy of NK cells was inhibited by 3-MA, resulting in significantly higher proliferation rates than those in control group. (E) Flow cytometry showed that NK cell function was significantly restored after its autophagy was inhibited. (F) Linear correlation analysis confirmed that there was a significant negative correlation between NK cell function and LC3 expression level. (G) Flow cytometry showed that under the influence of 3-MA, the number of lung-infiltrating NK cells didn’t show significant changes. (H) The RP+3-MA group significantly outperformed the RP-only group in terms of NK cell function. (A–H): n = 3. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. **P = 0.0058 (A), ***P < 0.0001 Ctrl vs RP+NAC, ***P < 0.0001 Ctrl vs RP, **P = 0.0019 RP vs RP+NAC (B), *P = 0.0253 72h, ***P < 0.0001 96h (D), *P = 0.0151 (E), ***P = 0.0004 IFN-γ, ***P = 0.0003 TNF-α (H).
Figure 5
Figure 5
Excessive autophagy leads to the internalization of NKG2D. (A) Using RT-PCR technology to examine RNA expression of the main functional receptors on the NK cell surface. And the reduced NKG2D expression on the surface of NK cells was further confirmed by western blotting (B) and flow cytometry (C). (D, E) Co-IP demonstrated a significant increase in the interaction between NKG2D and LAMP2 after radiation therapy, and inhibiting autophagy likewise attenuated their binding. (F) A reconstructed three-dimensional structured illumination microscopy (3D-SIM) image showing an NK cell. The cell surface is labeled with an antibody against NKG2D (green), and lysosomes are labeled with a specific lysosomal marker (red). The merged image demonstrates the co-localization of NKG2D and lysosomes (arrow) within the NK cell. Scale bar = 3 μm. A-E: n = 3. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. *P = 0.0236 NKp30, ***P = 0.0002 NKG2D, **P = 0.0044 NKG2A (A), ***P < 0.0001 (B), ***P = 0.0006 (C), **P = 0.0021, **P = 0.0016, AP2A1; **P = 0.0039, *P = 0.0390, AP2M1; **P = 0.0060, *P = 0.0472, LAMP2; ***P = 0.0009, *P = 0.0116, LC3 (E).
Figure 6
Figure 6
Autophagy-NK axis regulates radiation pneumonitis and tumor progression. (A) Animal model diagram. Evaluate the severity of radiation pneumonitis from three perspectives: HE of pulmonary tissue (B), and the number of neutrophils in bronchoalveolar lavage fluid (C). Then we examined the effect of autophagy and NK cells on tumor progression during radiotherapy using live animal imaging and survival time detection (D, E). (B, C): n = 3; (D, E): n = 5. Each point represents an individual experiment. *, P < 0.050; **, P < 0.010; ***, P < 0.001. *** P < 0.0001 Ctrl vs 3-MA, ***P = 0.0008 3-MA vs 3-MA+αNK1.1, ***P = 0.0005 3-MA+αNK1.1 vs αNK1.1 (B), **P = 0.0053 Ctrl vs αNK1.1, ***P = 0.0003 αNK1.1 vs 3-MA+αNK1.1, ***P < 0.0001 3-MA vs 3-MA+αNK1.1 (C), **P = 0.0065 Ctrl vs αNK1.1, ***P < 0.0001 αNK1.1 vs 3-MA+αNK1.1, *P = 0.0242, 3-MA+αNK1.1 vs 3-MA (E).
Figure 7
Figure 7
It presents a schematic diagram outlining the proposed mechanism. Key reactions, interactions, and resultant effects are represented with arrows. Radiation therapy induces DNA damage and cell death in lung tissue, leading to release of inflammatory cytokines like CXCL10. CXCL10 binds to and activates the CXCR3 receptor on natural killer (NK) cells, triggering signaling cascades involving RhoA and MAPK. This leads to recruitment and infiltration of more NK cells from circulation into the damaged lung tissue. Meanwhile, radiation also causes accumulation of reactive oxygen species (ROS) in lung tissue and NK cells. Excessive ROS induces overactivation of autophagy pathways in NK cells. This involves increased expression of autophagy proteins like LC3. Through adaptor proteins like AP2, LC3 binds to and internalizes the NKG2D activating receptor on NK cells, routing it for degradation in lysosomes. Loss of surface NKG2D impairs NK cell cytotoxicity and cytokine production, preventing efficient clearance of damaged cells and resolution of inflammation. Therefore, the CXCL10-CXCR3 axis and ROS-induced autophagy combine to both increase total NK cells yet suppress their functions in radiation pneumonitis.
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This study was funded by National Key Research and Development Program (2021YFC2500904, 2021YFC2500905), Young Taishan Scholars Program of Shandong Province (tsqnz20221167), Shandong Provincial Natural Science Foundation (ZR2022QH380, ZR2021LSW006), National Natural Science Foundation of China (82300193, 82303700), and China Postdoctoral Science Foundation (2022M721956, 2023TQ0200).