Research ArticleOriginal Article
Open Access

Osimertinib exacerbates immune checkpoint inhibitor-related severe adverse events by activating the IL-6/JAK/STAT3 pathway in macrophages

Yuan Li, Yanping Chen, Yuan Meng, Meng Shen, Fan Yang and Xiubao Ren
Cancer Biology & Medicine December 2024, 21 (12) 1156-1170; DOI: https://doi.org/10.20892/j.issn.2095-3941.2024.0269

Abstract

Objective: The combination of epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and immune checkpoint inhibitors (ICIs) leads to an increased incidence of severe immune-related adverse events (irAEs). However, the mechanisms underlying macrophages in irAEs have not been elucidated.

Methods: An osimertinib and ICI-induced irAE mouse model was constructed. Lung micro-CT scans were used to assess the degree of inflammatory infiltration. Hematoxylin-eosin staining was used to analyze the histopathologic inflammatory infiltration in mouse liver and lung tissues. Flow cytometry was used to detect the percentages of T cells, NK cells, and macrophages and the expression of EGFR. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum interleukin (IL)-6, alanine transaminase (ALT), ferritin, and tumor necrosis factor (TNF)-α levels. Total RNA extracted from mouse liver macrophages was analyzed by RNA-seq. Simple Western blot analysis was used to detect the IL-6/JAK/STAT3 pathway activation state.

Results: Osimertinib combined with ICIs upregulated EGFR expression on macrophages with increased serum IL-6, ALT, and ferritin levels. RNA-seq and simple Western blot analysis of mouse liver macrophages confirmed that that the IL-6/JAK/STAT3 pathway was activated in the combination treatment group. Ruxolitinib blocked the IL-6/JAK/STAT3 pathway and significantly decreased the serum IL-6, ALT, and ferritin levels in the combination treatment group.

Conclusions: An osimertinib and ICI-induced irAE mouse model was constructed that showed osimertinib combined with ICIs inhibited EGFR phosphorylation and activated the IL-6/JAK/STAT3 signaling pathway in mouse liver macrophages, which led to the release of relevant cytokines.

keywords

Introduction

Lung cancer is one of the most common and fatal malignant tumors worldwide, of which non-small cell lung cancer (NSCLC) accounts for 80%–85%1. The epidermal growth factor receptor (EGFR) mutation, which is mainly located in the kinase domain from exons 18–21, is one of the most common genetic mutations in patients with NSCLC and it is an important target for NSCLC therapy2. EGFR tyrosine kinase inhibitors (TKIs) are the standard treatment for patients with EGFR-mutant (Ex19del, L858R, and T790M mutations) NSCLC because the mutations significantly improve the remission and progression-free survival rates in EGFR-mutant NSCLC patients3. However, there is an urgent need for new and more effective treatments to overcome the limitation of EGFR-TKI resistance4.

Immunotherapy is another important treatment modality for NSCLC5. Immune checkpoint inhibitors (ICIs), such as PD-(L)1 inhibitors, are associated with durable responses in a proportion of patients with EGFR-mutant NSCLC6. EGFR-TKIs modulate the tumor immune microenvironment by inhibiting EGFR signal transduction, thereby boosting the antitumor activity of immunotherapy and increasing the proportion of patients benefiting from ICIs7–9. Therefore, clinicians are increasingly interested in EGFR-TKIs combined with ICIs for the treatment of EGFR-mutant advanced NSCLC. Immune-related adverse events (irAEs) are caused by overactivation of the immune system, which manifested as multi-organ inflammation and was associated with immune mechanisms in antineoplastic/therapeutic clinical trials. This “immune enhancement” strategy often resulted in rare objective responses and a high frequency of irAEs10. Several clinical trials, such as the phase Ib TATTON study (NCT02143466) and the phase III CAURAL trial (NCT02454933), terminated trial recruitment due to the increased incidence and severity of interstitial pneumonia caused by the combination of the two treatments11–13. However, the mechanisms responsible for the increased incidence of these severe adverse events are unclear.

Macrophages express EGFR. Numerous macrophages populate multiple organ injuries related to immunotherapy, but the mechanism is not clear14,15. Our preliminary animal experiments demonstrated that eliminating macrophages in mice significantly alleviated irAEs in vivo. Additionally, we used flow cytometry to detect wild-type EGFR expression on the surface of macrophages in the lung and liver tissues of mice with irAEs. It has also been shown that macrophages express PD-(L)1 and activation of EGFR on the macrophage surface can affect cytokine production16–18. Therefore, an osimertinib and ICI-induced irAE mouse model was constructed to determine the role of macrophages in irAEs, which would provide a possible theoretical basis for clinical research.

Materials and methods

Experimental animals

All animal experiments were approved by the Laboratory Animal Ethics Committee of Tianjin Medical University Cancer Institute & Hospital (Approval No. 2023057). Female BALB/c mice, 6–8 weeks old, were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) and fed adaptively for 7 days in a specific pathogen-free environment. The experimental animal sample per group design was based on preliminary experiments, the existing literature, and the “3R” animal ethics principle19,20.

Mouse model construction

Osimertinib (AZD9291) and ruxolitinib (INCB018424) were purchased from Selleck Chemicals (Shanghai, China) and dissolved at concentrations of 5 and 100 mg/kg in 0.5% CMC-Na (Selleck, Shanghai, China) and mice were gavaged once daily. Anti-mouse PD-1 (InVivoMAb anti-mouse PD-1, Clone RMP1-14; Bio X Cell, Lebanon, NH, USA) and anti-mouse CTLA-4 antibodies (InVivoMAb anti-mouse CTLA-4, Clone 9D9; Bio X Cell, Lebanon, NH, USA) were injected intraperitoneally (200 and 250 μg/dose, respectively) every 3 days. Control mice were administered equal amounts of 0.5% CMC-Na by gavage and/or equal amounts of isotype control immunoglobulin (Ig) by intraperitoneal injection. In the first cohort, mice were randomized into four groups of six mice each, as follows: (a) control group (PBS); (b) osimertinib group (TKI); (c) anti-PD-1 + anti-CTLA-4 group (immunotherapy group); and (d) osimertinib + anti-PD-1 + anti-CTLA-4 group (combination treatment group). In the second cohort, mice were randomly allocated to four groups of six mice each, as follows: (a) osimertinib + anti-PD-1 + anti-CTLA-4 group (TPC); (b) osimertinib + anti-PD-1 + anti-CTLA-4 + ruxolitinib group (TPCR); (c) anti-PD-1 + anti-CTLA-4 group (PC); and (d) anti-PD-1 + anti-CTLA-4 + ruxolitinib group (PCR).

Experimental methods

Mouse micro-CT imaging and serum enzyme-linked immunosorbent assay (ELISA)

Mice in the first cohort underwent low-dose CT scanning after inhalation of 2% isoflurane anesthesia on the 15th day of treatment. Blood was collected from the retro-orbital plexus, centrifuged, and the supernatant serum was collected and stored at −80°C for later use. Serum interleukin (IL)-6, alanine transaminase (ALT), tumor necrosis factor (TNF)-α, and ferritin levels were measured by ELISA with 2 sub-wells set up for each sample. The ELISA kits were obtained from Jianglai Biotech (Shanghai, China).

Flow cytometry of liver and lung tissues

After the mice were sacrifices, liver and lung tissues were digested with DNase I (Solarbio, Shanghai, China) and collagenase IV (Solarbio, Shanghai, China) to create single-cell suspensions. Flow cytometry was performed to detect the percentages of T cells (CD45+CD3+), NK cells (CD45+CD3-CD49b+), and macrophages (F4/80+CD11b+), as well as the expression of EGFR on T cells, NK cells, and macrophages in liver and lung tissues. F4/80 (#123114), CD11b (#101206), anti-mouse CD16/32 antibodies (#101320), and the Zombie NIR™ Fixable Viability kit (#423105) were acquired from Biolegend (San Diego, CA, USA). CD3 (#563024), CD45 (#550994), and CD49b antibodies (#560628) were obtained from BD Pharmingen (Milpitas, CA, USA). EGFR antibody (#48685S) was purchased from Cell Signaling Technology (Danvers, MA, USA). Data analysis was performed using FlowJo software (TreeStar, Inc., Franklin Lakes, NJ, USA).

Hepatic macrophage RNA-seq and simple Western blot

Mouse liver macrophages were enriched with Miltenyi Anti-F4/80 MicroBeads (#130-110-443; Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. TRIzol (Beyotime Biotechnology, Shanghai, China) was used to extract total RNA of mouse liver macrophages, which was sequenced and analyzed after completion of RNA quality control at Novogene (Beijing, China). A Padj < 0.05 and |log2-fold change| ≥ 0 were used as the screening thresholds for differentially expressed genes (DEGs). Software was used to analyze DEGs for Gene Ontology (GO) categories, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and Gene Set Enrichment Analysis (GSEA).

Liver macrophages were lysed in RIPA lysis buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitors (Absin, Shanghai, China). Protein concentrations were determined using the bicinchoninic acid (BCA) method. Janus-activated kinase (JAK) 1, JAK2, signal transducer and activator of transcription (STAT) 3, EGFR, and corresponding phosphorylation levels were evaluated using simple Western blot analysis in a Jess™ apparatus per the manufacturer’s protocol (ProteinSimple; Bio-techne, San Jose, CA, USA). The antibodies were purchased from Cell Signaling Technology. Blot analysis was performed on Compass for SW software (ProteinSimple). The protein level was calculated by the area under the chemiluminescence peak and normalized by the total capillary chemiluminescence of the given sample using the 12–230 kDa Separation Module.

Histopathologic analysis of liver and lung tissues

Parts of the liver and lung tissues from each mouse were fixed in 10% neutral buffered formalin, dehydrated, routinely embedded in paraffin, sectioned at a thickness of 4 μm, and stained with hematoxylin and eosin (HE) (Beyotime Biotechnology). Two pathologists randomly observed five fields of each tissue section under an optical microscope and scored the fields according to previously established criteria21,22.

Statistical analysis

GraphPad Prism software (version 9.0; GraphPad Software, Inc., Boston, MA, USA) was used for plotting and statistical analysis. Numerical results were tested for a normal distribution using the Shapiro–Wilk normality test and expressed as the mean ± standard deviation. The significance of the difference between two independent samples was determined using unpaired Student t-tests. One-way analysis of variance (ANOVA) was used for multiple comparisons among treatment groups. The Bonferroni method was used for multiple comparisons within groups. A P < 0.05 was considered to indicate a statistically significant difference.

Results

Construction of the irAE mouse model

Four groups of mice were treated in different ways for 15 days in the 1st cohort, (Figure 1A) and the inflammatory infiltration of the lungs was evaluated by micro-CT imaging (Figure 1B). The lung CT images of mice in the combination treatment and immunotherapy groups had different degrees of pleural effusions (indicated by arrows), whereas no obvious pleural effusion was seen in the control or osimertinib group (PBS represents the control group, TKI represents the osimertinib group, PC represents the immunotherapy group, and TPC represents the combination treatment group).

Figure 1
Figure 1
Figure 1

Construction of the irAE mouse model. (A) Mouse drug administration regimens in experimental cohort 1. (B) Micro-CT imaging was performed to evaluate pulmonary inflammatory infiltration in mice and the pleural effusion is indicated by an arrow. (C) Representative images of HE staining (scale bars, 100 and 50 μm). Lung tissue: alveolar edema (yellow arrow), alveolar and interstitial inflammation (red arrow), alveolar and interstitial hemorrhage (black arrow), atelectasis (white arrow), and necrosis and hyaline membrane formation (orange arrow); liver tissue: interfacial hepatitis (red arrow), portal inflammation (white arrow), and lytic necrosis and fusion necrosis of hepatocytes (yellow arrow). (D) Quantification of inflammatory pathology grading of lung and liver tissues of mice. All data are expressed as the mean ± standard deviation. Statistical comparison of treatment groups was performed using one-way ANOVA with the Bonferroni test. **P < 0.01, ***P < 0.001; P > 0.05 is not statistically significant (ns).

HE-stained mouse lung tissues under the microscope revealed distinct alveolar and interstitial hemorrhage, inflammatory pulmonary infiltrates, cell necrosis, and hyaline membrane formation (annotated by arrows) in the combination treatment and immunotherapy groups [Figure 1C (lung)]. Similarly, microscopic examination of HE-stained mouse liver tissues showed significant portal inflammation, as well as focal lytic and fusion necrosis of hepatocytes (marked by arrows) in the combination treatment and immunotherapy groups [Figure 1C (liver)]. The degree of pathologic inflammatory infiltration in liver and lung tissues was scored separately, according to previous studies21,22. The inflammatory scores in the combination treatment and immunotherapy groups were significantly higher than the control and osimertinib groups. Although there was a tendency for higher inflammation scores in the combination treatment group compared to the immunotherapy group, this difference was not statistically significant (Figure 1D). The imaging and pathologic findings indicated that ICIs combined with osimertinib successfully established an irAE mouse model.

The irAE mouse model has high levels of macrophages and T cells in liver and lung tissues

Flow cytometry was performed to determine the percentages of T cells, NK cells, and macrophages, as well as the expression of EGFR in liver and lung tissues. The percentage of T cells and macrophages in liver and lung tissues was significantly higher in the combination treatment and immunotherapy groups compared to the other groups. Although there was a trend towards a higher percentage of macrophages and T cells in the combination treatment group than the immunotherapy group, this difference did not reach statistical significance. The percentage of NK cells was highest in the combination treatment group liver tissues, whereas the opposite trend was observed in the lung tissues (Figure 2A and B). Additionally, EGFR expression on macrophages was a unique feature in the liver and lung tissues of mice within the TPC and PC groups. EGFR expression was not detected on T or NK cells (Figure 2C). The gating strategy for flow cytometry was illustrated in Figure S1A.

Figure 2
Figure 2

The irAE mouse model has high levels of macrophages and T cells in liver and lung tissues. (A) Flow cytometry results of macrophages from liver and lung tissues in different intervention groups. (B) Percentages of T cells, NK cells, and macrophages in lung (up) and liver (down) tissues in each group. (C) EGFR expression on T cells, NK cells, and macrophages. Results are presented as the mean ± standard deviation; P values were calculated using one-way ANOVA with the Bonferroni test; *P < 0.05, **P < 0.01, ***P < 0.001; P > 0.05 is indicated as no significance (ns).

Osimertinib combined with ICIs upregulated EGFR expression on macrophages and augmented the release of relevant cytokines

EGFR expression on macrophages varied significantly among the different subgroups of mouse tissues. The average EGFR fluorescence intensity on the surface of macrophages in the liver and lung tissues of mice was highest in the combination treatment group (TPC; Figure 3A). EGFR protein expression in mouse liver macrophages was higher in the combination treatment group compared to the other groups, as shown in the simple Western blot analysis (Figure 3B).

Figure 3
Figure 3

Osimertinib combined with ICIs increases EGFR expression on macrophages and the release of related cytokines. (A) EGFR expression on macrophages in different subgroups of mouse tissues. (B) Simple western blot results of EGFR in liver macrophages of mice in each group. (C) Serum IL-6, TNF-α, ALT, and ferritin levels were measured by ELISA in each group of mice. All data are expressed as the mean ± standard deviation; P values were calculated using one-way ANOVA with the Bonferroni test; *P < 0.05, **P < 0.01, ***P < 0.001; P > 0.05 is indicated as no significance (ns).

The serum IL-6, ALT, ferritin, and TNF-α levels were measured by ELISA. Serum IL-6, ALT, and ferritin levels in the combination treatment group were significantly higher than the other groups. TNF-α levels were higher in the combination treatment and immunotherapy groups but there was no significant difference between the two groups (Figure 3C). Taken together, these results suggested that osimertinib combined with ICIs upregulates EGFR expression on macrophages and augments the release of relevant cytokines.

The IL-6/JAK/STAT3 signaling pathway was enriched in mouse liver macrophages from the combination treatment group

The RNA-seq results of mouse liver macrophages in the combination treatment group (TPC) revealed a total of 4,209 DEGs, of which 1,776 genes were upregulated compared to the immunotherapy group (PC; Figure 4A and B). GO analysis identified 14 biological processes (BPs), 8 cellular components (CCs), and 8 molecular functions (MFs) that were associated with macrophage and cytokine secretion (Figure 4C).

KEGG enrichment analysis was performed on the 4,209 DEGs; 89 KEGG metabolic pathways were enriched. In addition to the pathways related to the metabolism of the three major substances and disease genesis, most of the remaining signaling pathways were associated with inflammatory responses and cytokines (Figure 4D), PPAR signaling pathway, C-type lectin receptor signaling pathway, Calcium signaling pathway, Th1 and Th2 cell differentiation, TNF signaling pathway, cGMP-PKG signaling pathway, MAPK signaling pathway. GSEA showed that the IL-6/JAK/STAT3 signaling pathway was significantly enriched in the combination treatment group compared to the immunotherapy group (Figure 4E and F).

Figure 4
Figure 4
Figure 4

The IL-6/JAK/STAT3 signaling pathway was notably enriched in mouse liver macrophages from the combination treatment group. (A–D) Volcano distribution map, heatmap of DEGs, GO, and KEGG enrichment analysis results (the combined intervention group vs. the immunotherapy group). (E and F) GSEA enrichment results of the combined intervention group compared with the immunotherapy group.

The IL-6/JAK/STAT3 signaling pathway was activated in mouse liver macrophages in the combination treatment group

The activation status of the IL-6/JAK/STAT3 signaling pathway and EGFR expression were verified by simple Western blot analysis and protein normalization, respectively. As shown by the blot bands, the p-JAK1, p-JAK2, and p-STAT3 levels were elevated in the combination treatment group compared to the other groups, while p-EGFR levels were suppressed (Figure 5).

Figure 5
Figure 5

The IL-6/JAK/STAT3 signaling pathway was activated in mouse liver macrophages in the combination treatment group. JAK1, JAK2, STAT3, and EGFR protein expression and phosphorylation levels in different intervention groups.

Ruxolitinib alleviated the release of relevant cytokines in the irAE mouse model

The specific dosing regimen for the second cohort is presented in Figure 6A. After 15 days of intervention, a pathologic analysis of the liver and lung tissues was performed. A microscopic view of HE-stained mouse lung tissues [Figure 6B (lung)] revealed varying degrees of alveolar cavity and interstitial hemorrhage, interstitial inflammatory infiltration, alveolar epithelial cell necrosis, and hyaline membrane formation in the four groups. Similarly, the microscopic view of HE-stained mouse liver tissues [Figure 6B (liver)] showed obvious portal area inflammation and varying degrees of hepatocyte necrosis in the experimental and control groups. Distinct scoring criteria were used to assess the degree of inflammatory infiltration in liver and lung tissues following HE staining (Figure 6C). There was no significant difference in inflammatory pathologic scores between the experimental (TPCR and PCR) and control groups (TPC and PC).

Figure 6
Figure 6
Figure 6

Ruxolitinib alleviated the release of relevant cytokines in the irAE mouse model. (A) Mouse drug administration regimens in experimental cohort 2. (B) Representative images of HE staining (scale bars, 100 and 50 μm). Lung tissue: alveolar edema (yellow arrow), alveolar and interstitial inflammation (red arrow), alveolar and interstitial hemorrhage (black arrow), atelectasis (white arrow), and necrosis and hyaline membrane formation (orange arrow); liver tissue: interfacial hepatitis (red arrow), portal inflammation (white arrow), and lytic necrosis and fusion necrosis of hepatocytes (yellow arrow). (C) Quantification of inflammatory pathology grading. (D) The serum IL-6, ALT, TNF-α, and ferritin levels in different intervention groups. Data are expressed as the mean ± standard deviation; P values were calculated using an unpaired Student t-test; **P < 0.01, ***P < 0.001; P > 0.05 is indicated as no significance (ns).

Whether blocking the IL-6 pathway could alleviate the release of relevant cytokines was then determined. The serum IL-6, ALT, and ferritin levels decreased significantly in the experimental group after the addition of ruxolitinib compared to the control group. The decrease was even more prominent in the combination treatment group with the addition of ruxolitinib (Figure 6D). However, there was no statistically significant difference in the TNF-α levels between the experimental and control groups. The above findings suggested that ruxolitinib reduced the release of serum-related cytokines induced by the combination of osimertinib and ICIs.

Discussion

There is strong evidence that combination therapies can overcome or delay drug resistance in EGFR-mutant NSCLC. There has recently been a growing interest in EGFR-TKIs combined with ICIs for the treatment of EGFR-mutant advanced NSCLC. However, several clinical trials were discontinued due to the increased incidence and severity of irAEs11–13. Therefore, there is an urgent need to explore possible mechanisms underlying irAEs and propose feasible solutions. In the current study an irAE mouse model was constructed using osimertinib, an irreversible third-generation EGFR-TKI23, combined with ICIs. Although osimertinib has a relatively weak affinity for wild-type EGFR (IC50, 184 nM), osimertinib still exhibits an inhibitory effect24. Only osimertinib combined with ICIs was studied because previous studies have shown that irAEs produced by this combination are specific for osimertinib and osimertinib monotherapy is well-tolerated, suggesting that osimertinib is well-suited for combination therapy20,25. The current study revealed one possible mechanism for the increased incidence and severity of irAEs caused by osimertinib combined with ICIs, which would provide a necessary theoretical basis for future clinical treatment.

Currently, no definitive biomarkers exist to differentiate between irAEs and immune activation. The potential predictive value of serum chemokines (e.g., CXC), cytokines (IL-6, IL-8, IL-10, and IL-17), ferritin, and CRP has been suggested26–30. The immunotherapy-associated pneumonia pathology is characterized by the presence of numerous macrophages and vacuolization of alveolar epithelial cells under the microscope31, with a significant increase in CD8+ T cells and M1 macrophages in the immune microenvironment, a decrease in resting NK cells, higher activity of inflammatory response pathways, and lower activity of immune depletion-related pathways32. A similar situation has been reported in immunotherapy-associated hepatitis and myocarditis14,15,33. The current study revealed that serum IL-6, ALT, and ferritin levels were significantly higher in the combination treatment group compared to the immunotherapy group but there were no significant differences in histopathologic inflammation scores or macrophage percentages in liver or lung tissue between the two groups. A validation experiment was also conducted in BALB/c mice with subcutaneous tumors (4T1), and the results from both experiments were consistent (Figure S1B).

We attempted to determine the reason for the inconsistency between serologic and pathologic results. Macrophages are the primary cellular source of mouse serum ferritin34 and ferritin can be used as a marker for the diagnosis, differential diagnosis, and prognosis of irAEs30. The increase in mouse serum ferritin further supports the crucial role that macrophages might play in irAEs. Activation of EGFR on the surface of macrophages can affect the production of cytokines17,35,36. Our results confirmed that mouse liver and lung macrophages express EGFR and osimertinib combined with ICIs upregulate the expression of EGFR on macrophages. Therefore, whether macrophages have a significant role in the irAE mouse model induced by osimertinib combined with ICIs was investigated, which could help explain the inconsistency in the results.

The main mechanism underlying EGFR-TKIs is that EGFR-TKIs bind to the intracellular tyrosine kinase domain of EGFR, which affects the phosphorylation of tyrosine residues and inhibits major signaling pathways of EGFR, such as the Ras/Raf/MEK/ERK-MAPK and PI3K/Akt/mTOR pathways37. ICIs directly or indirectly act on targets, such as PD-(L)1 and B7m on the surface of macrophages38, which causes alterations in the activation state of some intracellular signaling pathways. JAK and STAT subtypes (mainly JAK2/STAT3) are upregulated in patients with interstitial lung disease (ILD) and animal models, while upregulated cytokines and growth factors in ILD activate this signaling pathway39. RNA-seq was performed on mouse liver macrophages and it was shown that the IL-6/JAK/STAT3 signaling pathway was significantly enriched in the combination treatment group. Simple Western blot analysis confirmed that the pathway was activated. In subsequent experiments a dual-approach strategy was used with immunofluorescence to visualize the expression patterns of p-EGFR within macrophages, the results of which were consistent with the quantitative insights provided by simple Western blot analysis. This integrated approach has the potential to significantly bolster the credibility and persuasiveness of our experimental findings. In summary, we can discern a plausible explanation for the discrepancy observed between histopathologic and serologic outcomes. In mice with irAEs, EGFR is expressed on the surface of liver macrophages, which also harbor immunosuppressive receptors. When osimertinib is administered in combination with ICIs, the phosphorylation of EGFR (wild-type) on the surface of liver macrophages is inhibited in mice and the IL-6/JAK/STAT3 signaling pathway is activated, leading to an increase in the release of cytokines in the serum, resembling a “cytokine storm.” This potential mechanism provides a compelling explanation for the inconsistencies observed in our experiments (Figure 7).

Figure 7
Figure 7

Schematic diagram of the IL-6/JAK/STAT3 signaling pathway activation in mouse liver macrophages. IRs, inhibitory receptors (IRs); ICIs, immune checkpoint inhibitors; IL-6, interleukin-6; ALT, alanine transaminase (ALT). In mice with immune-related adverse events (irAEs), EGFR is expressed on the surface of liver macrophages, which also harbor immunosuppressive receptors. When osimertinib is administered in combination with immune checkpoint inhibitors (ICIs), phosphorylation of EGFR (wild-type) on the surface of liver macrophages is inhibited and the IL-6/JAK/STAT3 signaling pathway is activated, leading to an increase in the release of cytokines into the serum, resembling a “cytokine storm.” Notably, the addition of ruxolitinib, a small molecule inhibitor of JAK1/JAK2, to block the IL-6/JAK/STAT3 signaling pathway resulted in a significant decrease in the levels of these cytokines in the serum.

Blocking the IL-6 pathway could eliminate immunotherapy toxicity and promote tumor immunity40. Ruxolitinib, a JAK1/JAK2 inhibitor with a variety of anti-inflammatory and immune-enhancing efficacy41, was added to the combination treatment and immunotherapy groups. The serum IL-6, ALT, and ferritin levels were significantly reduced after the addition of ruxolitinib in the combination treatment and immunotherapy groups but the combination treatment group was more significantly decreased. However, compared to the control groups, the experimental groups showed no improvement in pathologic injury and inflammatory infiltration in liver and lung tissues. These results indicated that osimertinib combined with ICIs inhibited EGFR phosphorylation and activated the IL-6/JAK/STAT3 signaling pathway in mouse liver macrophages. Ruxolitinib blocked this pathway, thus reducing the release of related cytokines. Moreover, the TNF-α levels were higher in the combination treatment and immunotherapy groups but the TNF-α level did not decrease significantly after the addition of ruxolitinib. A previous study showed that TNF-α is mainly produced by monocytes and macrophages42, which involves the NF-κB and c-Jun signaling pathways43. Therefore, the TNF-α level did not decrease after blocking the IL-6/JAK/STAT3 pathway. In corollary studies we will actively incorporate anti-IL-6 receptor44, dexamethasone, and other relevant agents into our intervention strategies to discover an efficient and optimized treatment regimen that can significantly reduce the occurrence and impact of irAEs. This approach will provide us with a new perspective on understanding the pathologic mechanisms underlying irAEs and drive progress in clinical treatment.

Conclusions

In summary, we constructed an irAE mouse model induced by osimertinib combined with ICIs and found that osimertinib combined with ICIs inhibited EGFR phosphorylation and activated the IL-6/JAK/STAT3 signaling pathway in mouse liver macrophages, leading to the release of relevant cytokines. This finding provides a possible theoretical basis for clinical research. The study had limitations. First, we only investigated the role played by macrophages in this model, so we need to determine whether other cells have a synergistic role with macrophages. Second, our study focused only on the irAEs caused by the combination of the two drugs without exploring their therapeutic efficacy. Third, our conclusions were based solely on preclinical animal model experiments and require further clinical validation.

Supporting Information

Conflict of interest statement

No potential conflicts of interest are disclosed.

Author contributions

Conceived and designed the analysis: Xiubao Ren, Fan Yang.

Collected the data: Yuan Li, Yanping Chen, Meng Shen.

Contributed data or analysis tools: Yuan Meng, Meng Shen.

Performed the analysis: Yuan Li, Yanping Chen, Fan Yang.

Wrote the paper: Yuan Li, Yanping Chen.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Footnotes

  • ↵*These authors contributed equally to this work.

  • Received July 17, 2024.
  • Accepted October 25, 2024.

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