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. 2024 Jul 17;17(7):e14522. doi: 10.1111/1751-7915.14522

Akkermansia muciniphila outer membrane protein regulates recruitment of CD8 + T cells in lung adenocarcinoma and through JAK–STAT signalling pathway

Yufen Xu 1, Xiaoli Tan 2, Qi Yang 2, Zhixian Fang 2, Wenyu Chen 2,✉
PMCID: PMC11253302  PMID: 39016683

Abstract

As a Gram‐negative anaerobic bacterium, Akkermansia muciniphila (AKK) participates in the immune response in many cancers. Our study focused on the factors and molecular mechanisms of AKK affecting immune escape in lung adenocarcinoma (LUAD). We cultured AKK bacteria, prepared AKK outer membrane protein Amuc_1100 and constructed a subcutaneous graft tumour mouse model. A549, NCI‐H1395 cells and mice were respectively treated with inactivated AKK, Amuc_1100, Ruxolitinib (JAK inhibitor) and RO8191 (JAK activator). CD8+ T cells that penetrated the membrane were counted in the Transwell assay. The toxicity of CD8+ T cells was evaluated by lactate dehydrogenase assay. Western blot was applied to determine JAK/STAT‐related protein and PD‐L1 expression, whilst CCL5, granzyme B and INF‐γ expression were assessed through enzyme‐linked immunosorbent assay (ELISA). The proportion of tumour‐infiltrating CD8+ T cells and the levels of granzyme B and INF‐γ were determined by flow cytometry. AKK markedly accelerated A549 and NCI‐H1395 recruiting CD8+ T cells and enhanced CD8+ T cell toxicity. Amuc_1100 purified from AKK exerted the same promoting effects. Besides, Amuc_1100 dramatically suppressed PD‐L1, p‐STAT and p‐JAK expression and enhanced CCL5, granzyme B and INF‐γ expression. Treatment with Ruxolitinib accelerated A549 and NCI‐H1395 cells recruiting CD8+ T cells, enhanced CD8+ T cell toxicity, CCL5, granzyme B and INF‐γ expression, and inhibited PD‐L1 expression. In contrast, the RO8191 treatment slowed down the changes induced by Amuc_1100. Animal experiments showed that Amuc_1100 was found to increase the number of tumour‐infiltrating CD8+ T cells, increase the levels of granzyme B and INF‐γ and significantly inhibit the expression of PD‐L1, p‐STAT and p‐JAK, which exerted an antitumour effect in vivo. In conclusion, through inhibiting the JAK/STAT signalling pathway, AKK outer membrane protein facilitated the recruitment of CD8+ T cells in LUAD and suppressed the immune escape of cells.

AKK promoted the recruitment of CD8+ T cells by lung adenocarcinoma (LUAD) cells and the toxicity of CD8+ T cells; AKK outer membrane protein was the main substance involved in the immune escape of LUAD. Through the JAK/STAT signalling pathway, AKK outer membrane protein promoted the recruitment of CD8+ T cells by LUAD cells and toxicity of CD8+ T cells, and inhibited immune escape.

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INTRODUCTION

Lung cancer stands as one of the most common malignancies worldwide, featuring the fastest growing in both incidence and mortality rates. This cancer encompasses two primary histological subtypes: small‐cell lung cancer and non‐small cell lung cancer (NSCLC) (Oliver, 2022). Amongst these, lung adenocarcinoma (LUAD) is the most predominant histological subtype of NSCLC. It is invasive and fatal, which covers approximately 50% of lung cancers (Zhu et al., 2022). Complex interactions of genetic, dietary, lifestyle and environmental factors occur in LUAD genesis and progression. Association between microflora and malignant progression of lung cancer was previously reported (Pizzo et al., 2022). On the one hand, flora can promote tumour progression. For example, commensal bacteria promote bone marrow cells to produce IL‐23 and IL‐1β, accelerate Vgamma6+ Vdelta1+ γδ T cells activation and proliferation that generates molecules including IL‐17, thereby enhancing tumour cell proliferation and inflammation (Jin et al., 2019). On the other hand, beneficial bacteria in combination with drugs can improve treatment outcomes. For example, Clostridium butyricum markedly prolonged progression‐free survival and overall survival (OS) in NSCLC patients treated with immune checkpoint blockade (Tomita et al., 2020). Therefore, exploring possible beneficial bacteria helps to understand the association between microorganisms and LUAD and improve the outcome of LUAD patients.

As a Gram‐negative anaerobic bacterium, Akkermansia muciniphila (AKK) exists in the intestinal mucus layer and covers 1–4% of the total faecal bacterial count in healthy adults (Guo et al., 2017). Recent studies reported that as a probiotic, AKK is valuable in improving immune response and host metabolic function (Zhang et al., 2019). Plovier et al. (2017) demonstrated that after pasteurisation, AKK‐purified outer membrane protein Amuc_1100 remained stable, which improved metabolism in obese diabetic mice. Ansaldo et al. (2019) illustrated that AKK regulated host immune function during homeostasis in vivo by inducing IgG antibody and antigen‐specific T‐cell responses. Wang et al. (2020) verified that Amuc_1100 suppressed colitis‐related tumorigenesis by enhancing CD8+ T cell immune infiltration in mice. The role of AKK in lung cancer was previously explored by researchers such as Derosa et al. (2022) who demonstrated that the faecal AKK abundance in NSCLC patients treated with PD‐L1 was associated with their increased objective response rates and OS. Chen et al. (2020) found that for lung cancer mice, treatment with cisplatin and AKK markedly attenuated tumour volume growth, increased TNF‐α, IL‐6 and IFN‐γ expressions and inhibited CD4+ CD25+ Foxp3+ Treg expression in peripheral blood and spleen of mice. Their study suggested that treatment of cisplatin and AKK may enhance the antitumor effect of cisplatin. Nonetheless, the factors and molecular mechanisms through which AKK impacts immune escape in LUAD have yet to be fully elucidated. Thus, further investigation is imperative to delve into the intricate mechanism of AKK influencing the immune microenvironment in LUAD.

This study unveiled that AKK accelerated the recruitment of CD8+ T cells by A549 cells and enhanced the toxicity of CD8+ T cells. As the main substance influencing immune regulation of CD8+ T cells, Amuc_1100 was found to inhibit proteins with JAK/STAT signalling pathway whilst simultaneously elevating the expression of T cell chemokine CCL5. Additionally, the JAK inhibitor Ruxolitinib accelerated the recruitment of CD8+ T cells by A549 cells, increasing the toxicity of CD8+ T cells and inhibiting the immune escape of the cells. At the same time, we demonstrated the antitumor effects of AKK in vivo. Our study highlighted that AKK affected LUAD through the involvement of Amuc_1100 in immune escape by regulating CD8+ T cell toxicity. This study confirmed AKK as a potential therapeutic target for LUAD patients, offering a new theoretical basis for their clinical treatment.

EXPERIMENTAL PROCEDURES

AKK incubation and pasteurization

AKK was cultured in brain heart infusion broth with 10 mg/L Resazurin (American Type Culture Collection, USA), a redox indicator, in an anaerobic environment. Mucin media with 1% agarose was applied for counting. Colony forming units (CFU) per mL in the anaerobic environment were determined using a representative medium. We used anaerobic PBS with 2.5% glycerol for diluting the medium to 1.5 × 108 CFU/μL and then pasteurized them (70°C, 30 min) (Wang et al., 2020).

Preparation of AKK outer membrane protein Amuc_1100

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3). After expansion culture and induction, the cells were collected and subjected to high‐pressure disruption. The target protein was bound to Ni‐NTA using a low‐concentration imidazole solution and then eluted with a high‐concentration imidazole solution. The presence of the His‐tagged outer membrane protein Amuc_1100 was confirmed by protein blotting. Finally, the His‐tagged Amuc_1100 was further purified using a HiLoad 16/60 Superdex 200 column on an AKTA protein purification system. Coomassie Brilliant Blue staining showed successful in vitro recombination of Amuc_1100.

Cell selection and processing

Human LUAD cell lines A549 and NCI‐H1395 (ATCC, USA) were cultured in Roswell Park Memorial Institute‐1640 medium with 10% fetal bovine serum (FBS). We seeded the cells into 96‐well plates with AKK/Amuc_1100 (10 μg/mL)/His (200 μg/mL)/Ruxolitinib (diluted to 0.1–5000 nM in 1% dimethylsulfoxide (DMSO)) (Buker, Germany), 2 μM RO8191 (MedChemExpress, USA) or PBS/DMSO as the control and incubated them for 24 h for upcoming assays (Hu et al., 2014; Wang et al., 2020).

Western blot

Cells were lysed in a radioimmunoprecipitation assay buffer, whilst the concentration of proteins was detected by a BCA kit (Beyotime, China). The proteins were separated on SDS‐PAGE and shifted to polyvinylidene fluoride membranes. The membranes were blocked with skim milk and then cultured overnight with primary antibodies anti‐human PD‐L1 (1:1000, ab213524, Abcam, UK), anti‐mouse PD‐L1 (1:1000, ab213480, Abcam, UK), anti‐p‐STAT (1:1000, ab32143, Abcam, UK), anti‐STAT (1:1000, ab68153, Abcam, UK), anti‐JAK (1:5000, ab108596, Abcam, UK), anti‐p‐JAK (1:5000, ab32101, Abcam, UK) and GAPDH (1:1000, ab8245, Abcam, UK). Then the proteins were incubated with horseradish peroxidase‐labelled secondary antibody IgG (1:2000, ab6721, Abcam, UK). Protein bands were imaged using an electrochemiluminescence system.

Enzyme‐linked immunosorbent assay (ELISA)

IFN‐γ (KHC4021, Thermo Fisher Scientific, USA), granzyme B (BMS2027‐2, Thermo Fisher Scientific, USA) and CCL5 (EHRNTS, Thermo Fisher Scientific, USA) expressions in the supernatants of A549 and NCI‐H1395 cells with different treatments were determined using corresponding ELISA kits.

Transwell assay

CD8+ T cells penetrating the membrane were calculated referring to a previous study (Song et al., 2020). Differently treated A549 cell cultures and NCI‐H1395 cell cultures were added to the lower chamber, whilst CD8+ T cells were added to the upper chamber. After 4 h incubation, we collected the cells migrating to the lower chamber and counted them using a cell counter.

Lactate dehydrogenase (LDH) assay

According to a previous study (Man et al., 2016), we first co‐cultured differently treated A549 and NCI‐H1395 cells with CD8+ T cells for 48 h and performed an LDH assay to determine cytotoxicity. Optical density at 490 nm was detected after 1 h. Using the following formula, the proportion of cells killed was calculated:

Cytotoxicity%=test sample‐lowcontrol/highcontrol‐lowcontrol×100%.

Subcutaneous allograft tumour mouse model

Fifteen male C57BL/6 mice (6–8 weeks old, 20–23 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. and maintained under specific pathogen‐free (SPF) conditions. The animal protocol and experimental procedures were approved by the Ethics Committee and carried out following the guidelines for the care and use of laboratory animals set by the Ethics Committee.

For the subcutaneous inoculation procedure, 1 × 106 LLC cells were administered into the dorsal region of C57BL/6 mice. The mice were then randomly allocated into three groups (n = 5): the control group, the Amuc group and the Amuc+RO8191 (JAK activator) group. Intraperitoneal administration was executed with the control group receiving 0.9% saline, the Amuc group receiving Amuc (3 μg) and the Amuc+RO8191 group receiving a combination of Amuc (3 μg) and RO8191 (2 mg/kg) (Lin et al., 2022; Wang et al., 2020). The assessment of tumour volume commenced on day 8 post‐injection and continued at 3‐day intervals. The formula employed for the calculation of tumour volume was: tumour volume = (length × width2)/2. On day 20, the mice were euthanized, tumours were dissected and further experiments were conducted.

Flow cytometry analysis

Fresh mouse subcutaneous tumour tissues were minced into small pieces and digested at 37°C for 45 min in 1% FBS in PBS supplemented with Type VI Collagenase (2 mg/mL, Thermo Fisher Scientific, USA). During this process, the samples were agitated at a speed of 220 rpm.

The digested suspension was filtered through the 80 μm filter, followed by washing twice in FACS buffer. Then single‐cell suspensions were resuspended in FACS buffer for staining with the appropriate surface antibodies. The stained cells were subsequently analysed using flow cytometry (NovoCyte Advanteon, Agilent, USA). The antibodies utilized in the staining process included FITC anti‐mouse CD3 Antibody (100203), PerCP anti‐mouse CD8a Antibody (100731), APC anti‐mouse CD45 Antibody (147707), PE anti‐mouse IFN‐γ Antibody (163503), APC anti‐human/mouse Granzyme B Recombinant Antibody (372203), all of which were procured from Biolegend (USA).

Statistical analysis

All assays were done three times. We applied GraphPad Prism 8.0 (USA) for data analyses. Results are presented as mean ± standard deviation. We first used the Shapiro–Wilk test to assess the normality distribution of the data, and the analysis showed that it did not fit the normal distribution due to the small amount of data (N too small), so we used the non‐parametric Mann–Whitney test for comparisons between different two groups. The data did not involve comparisons between multiple groups, so there was no analysis of variance or multiple comparisons. p < 0.05 indicates statistically significant.

RESULTS

AKK regulates chemotaxis and killing ability of CD8 + T cells

To reveal the effect of AKK on A549 and NCI‐H1395 cell immunity, we cultured A549 and NCI‐H1395 cells in a medium with/without inactivation of AKK for 24 h. In the Transwell assay, we counted the CD8+ T cells penetrating the membrane, which suggested that AKK notably accelerated A549 and NCI‐H1395 cells recruiting CD8+ T cells (Figure 1A). We then co‐cultured the treated A549 and NCI‐H1395 cells with CD8+ T cells for LDH assay to determine CD8+ T cell toxicity, which showed that AKK markedly enhanced the killing ability of CD8+ T cells (Figure 1B). Subsequently, ELISA demonstrated that AKK remarkably elevated CCL5, granzyme B and INF‐γ expression in A549 and NCI‐H1395 cells (Figure 1C–E). The above results confirmed that AKK accelerated A549 and NCI‐H1395 cells recruiting CD8+ T cells and suppressed the immune escape of tumour cells.

FIGURE 1.

FIGURE 1

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AKK regulates chemotaxis and killing ability of CD8+ T cells. (A) Transwell assay for counting CD8+ T cells penetrating the membrane after AKK treatment. (B) LDH assay for detecting CD8+ T cell toxicity after AKK treatment. (C–E) ELISA for determining CCL5, granzyme B and IFN‐γ expression after AKK treatment. * indicates p < 0.05.

Amuc_1100 is the main substance in AKK that affects immune escape

To reveal the factors of AKK‐mediated immune escape of A549 and NCI‐H1395 cells, we purified Amuc_1100 from AKK. Firstly, we transformed the recombinant plasmid into E. coli BL21 for expression. After the construction of the recombinant plasmid was completed, the plasmid was subjected to MluI and XhoI enzyme digestion and analysed by 1% agarose gel electrophoresis. The observed bands matched the expected results, indicating that the plasmid was suitable for subsequent transformation (Figure 2A). Subsequently, the target protein Amuc_1100 was eluted using an imidazole solution and the Amuc_1100 with His‐tag was verified by western blot (Figure 2B). Finally, the Amuc_1100 with His‐tag was filtered and purified, and its successful synthesis in vitro was confirmed using Coomassie Brilliant Blue staining (Figure 2C).

FIGURE 2.

FIGURE 2

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Purification of AKK outer membrane protein Amuc_1100. (A) After the recombinant plasmid construction was completed, the plasmids were digested by MluI and XhoI and analysed by 1% agarose gel electrophoresis. (B) Western blot for verifying the successful expression of the outer membrane protein Amuc_1100 with His‐tag. (C) Coomassie Brilliant Blue staining for validating Amuc_1100 purification.

Next, A549 and NCI‐H1395 cells were treated with purified Amuc_1100. Transwell assay demonstrated that Amuc_1100 markedly accelerated A549 and NCI‐H1395 cells recruiting CD8+ T cells (Figure 3A). We co‐cultured the treated A549 and NCI‐H1395 cells with CD8+ T cells for LDH assay, which illustrated that Amuc_1100 notably enhanced the killing ability of CD8+ T cells (Figure 3B). ELISA confirmed that Amuc_1100 markedly elevated CCL5, granzyme B and INF‐γ expressions in A549 and NCI‐H1395 cells (Figure 3C–E). In addition, none of the experimental results in the His protein group differed significantly from those in the PBS control group, suggesting that the failure of Amuc_1100 to remove the His tag does not affect its function. Thus, it was suggested that Amuc_1100 was the main substance in AKK that affected immune escape.

FIGURE 3.

FIGURE 3

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AKK outer membrane protein Amuc_1100 regulates chemotaxis and killing ability of CD8+ T cells. (A) Transwell assay for determining CD8+ T cells penetrating the membrane after Amuc_1100 treatment. (B) LDH assay for evaluating CD8+ T cell toxicity after Amuc_1100 treatment. (C–E) ELISA for assessing CCL5, granzyme B and IFN‐γ expression after Amuc_1100 treatment. * indicates p < 0.05.

AKK outer membrane protein regulates CD8 + T cell recruitment and immune escape of A549 and NCI‐H1395 cells through inhibiting JAK–STAT signalling pathway

It was previously illustrated that improvement of the cisplatin treatment outcomes by AKK may be related to the JAK/STAT pathway (Chen et al., 2020). For intensive investigation on how AKK affected CD8+ T cells, we treated A549 and NCI‐H1395 cells with Amuc_1100 and RO8191 (JAK agonist) for western blot to assess the expressions of PD‐L1 on the surface of A549 and NCI‐H1395 cells and JAK/STAT pathway‐related proteins. We demonstrated that Amuc_1100 notably hindered p‐JAK, p‐STAT and PD‐L1 expressions, which were reversed by Amuc_1100 and RO8191 co‐treatment (Figure 4A,B). ELISA then demonstrated that Amuc_1100 elevated T‐cell chemokine CCL5 expression notably, which was reversed by Amuc_1100 and RO8191 co‐treatment (Figure 4C). To further investigate the regulatory role of the JAK/STAT signalling pathway on immunity, we treated cells with JAK inhibitor Ruxolitinib and performed the Transwell assay to evaluate CD8+ T cells penetrating the membrane. Ruxolitinib was verified to remarkedly accelerate the recruitment of CD8+ T cells by A549 and NCI‐H1395 cells (Figure 4D). We co‐cultured the treated cells with CD8+ T cells afterwards and performed the LDH assay, which demonstrated the promoting effect of Ruxolitinib on CD8+ T cell toxicity (Figure 4E). As the western blot assay suggested, Ruxolitinib lowered PD‐L1 expression in cells (Figure 4F,G), whilst elevating CCL5 expression by ELISA (Figure 4H). Therefore, we demonstrated that AKK accelerated cancer cells recruiting CD8+ T cells and inhibited immune escape by suppressing the JAK/STAT signalling pathway.

FIGURE 4.

FIGURE 4

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AKK outer membrane protein regulates CD8+ T cell recruitment and immune escape of A549 cells by inhibiting the JAK–STAT signalling pathway. (A, B) Western blot for determining JAK/p‐JAK, STAT/p‐STAT and PD‐L1 expressions after different treatments and their quantitative analysis. (C) ELISA for evaluating the level of T‐cell chemokine CCL5 after different treatments. (D) Transwell assay for calculating CD8+ T cells penetrating the membrane after Ruxolitinib treatment. (E) LDH assay for assessing the toxicity of CD8+ T cells after Ruxolitinib treatment. (F, G) Western blot for evaluating PD‐L1 expression after Ruxolitinib treatment and their quantitative analysis. (H) ELISA for detecting T‐cell chemokine CCL5 expression after Ruxolitinib treatment. * indicates p < 0.05.

AKK outer membrane protein inhibits tumour progression in LUAD mice by recruiting CD8 + T cells through inhibition of the JAK–STAT signalling pathway

To provide further insight into the immunological mechanism underlying the role of AKK in regulating CD8+ T cell‐mediated anti‐tumour activity, we stratified LUAD mice into three groups: PBS + DMSO group, Amuc_1100 + DMSO group and Amuc_1100 + ROB191 group. Compared to the PBS + DMSO group, the administration of Amuc_1100 yielded a significant inhibition of tumour growth, but the addition of RO8191 attenuated the inhibitory effect of Amuc_1100 on tumours (Figure 5A–C). We evaluated the infiltration of CD8+ T cells in mouse tumours using flow cytometry and found that Amuc_1100 significantly increased the proportion of tumour‐infiltrating CD8+ T cells, whilst RO8191 administration slowed down this trend (Figure 5D). Furthermore, Amuc_1100 markedly increased the expression of IFN‐γ and granzyme B in CD8+ T cells, but when Amuc_1100 and ROB191 were administered in combination, the levels of both factors showed no significant difference compared to the control group (Figure 5E). Finally, western blot analysis unveiled that Amuc_1100 exerted significant inhibitory effects on the expression of p‐JAK, p‐STAT and PD‐L1 in tumour tissue. Conversely, co‐administration of Amuc_1100 and RO8191 reversed the expression of p‐JAK, p‐STAT and PD‐L1 (Figure 5F). Collectively, these results indicated that AKK recruited CD8+ T cells and inhibited the progression of LUAD by suppressing the JAK–STAT signalling pathway in an in vivo context.

FIGURE 5.

FIGURE 5

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Akk outer membrane protein inhibits tumour progression in LUAD mice by recruiting CD8+ T cells through inhibition of the JAK–STAT signalling pathway. (A) Tumour growth curves of mice in each group. (B) Weight of tumours of mice in each group weighed after execution. (C) Images of tumour tissues of mice in each group. (D) Representative FACS images and quantification of intratumoral CD8+ T cells after different treatments. (E) Representative FACS images and quantification of IFN‐γ and Granzyme B in CD8+ T cells after different treatments. (F) Western blot for determining JAK/p‐JAK, STAT/p‐STAT and PD‐L1 expressions after different treatments. * indicates p < 0.05.

DISCUSSION

Lung cancer is the leading cause of cancer‐related deaths worldwide, and the gut microbiome plays an important role in the development of lung cancer (Zhao et al., 2021). The relationship and interactions between the gut and lungs are known as the gut–lung axis (GLA) (Zhang & Xu, 2023). For example, the lung flora can influence the gut flora through blood circulation (Renz et al., 2011), and the gut microbiota induces a variety of respiratory disorders such as COPD, respiratory infections and asthma (Bingula et al., 2017). The GLA immune interactions are a bi‐directional process stemming from multifaceted interactions, as well as local and distal immune effects. Changes in GLA may lead to deleterious outcomes such as cancer development, pathogen colonisation, tissue damage and increased susceptibility to infection (Zhao et al., 2021).

Increasingly studies have reported that particular intestinal commensal bacteria and metabolites can hinder tumorigenesis (Gao et al., 2017). As a crucial intestinal commensal bacterium, AKK is widely regarded as a potential next‐generation probiotic that may affect the immunotherapy efficacy and tumour progression through modulating tumour immune microenvironment (Dingemanse et al., 2015; Routy et al., 2018). Luo et al. (2021) demonstrated that AKK‐EV hinders the malignant progression of prostate cancer cells by elevating the number of M1‐like macrophages, IFN‐γ+ CD8+ and GZMB+ CD8+ T cells. Routy et al. (2018) found relatively high AKK levels in tumour patients who responded to anti‐PD‐1 immunotherapy. The efficacy of PD‐1 blockade is restored by AKK supplementation through accelerating the recruitment of CCR9+ CXCR3+ CD4+ T cells in mouse tumours in an interleukin 12‐dependent manner. In colorectal cancer, AKK inhibits tumour progression through enhancing M1‐like macrophage enrichment mediated by TLR2/NLRP3 (Fan et al., 2021). Our study demonstrated that AKK accelerated LUAD cells recruiting CD8+ T cells in vivo and in vitro environments and enhancing the killing ability of CD8+ T cells, which may work as a potential therapeutic target for LUAD.

It may be the cell surface components or metabolites that contribute to the positive effects of AKK. For instance, it was revealed that AKK recombinant protein Amuc_1434* activates the death receptor and mitochondrial apoptosis pathways through the up‐regulation of tumour‐necrosis‐factor‐related apoptosis‐inducing ligand, which suppresses colorectal cancer LS174T cell activity (Meng et al., 2020). Ottman et al. (2017) found that by activating Toll‐like receptors 2 and 4, purified Amuc_1100 protein and concentration with all its related proteins stimulate the generation of specific cytokines, thereby accelerating IL‐10 release and improving intestinal barrier function and host immune homeostasis in the intestinal mucosa. We illustrated that outer membrane protein Amuc_1100 accelerated LUAD cells recruiting CD8+ T cells and enhanced the killing ability of CD8+ T cells, which was the main substance of AKK that regulates immune escape in tumours.

We also revealed that Amuc_1100 suppressed cellular immune escape by inhibiting JAK/STAT pathway protein expressions. The relationship between immune regulation and JAK/STAT was previously investigated. Wang et al. (2021) reported that ARNTL2 may elevate infiltration levels of CD8+ T cells and CTLA4, PD1, PD‐L1 and PD‐L2 expressions by activating the JAK/STAT pathway, thereby enhancing immune escape of clear cell renal carcinoma cells. Ravindran Menon et al. (2021) illustrated that CD8+ T cells can be activated through suppressing PD‐L1 expression and JAK/STAT signalling pathway, thereby enhancing the antitumor immune response in melanoma. In the present study, we found that JAK inhibitor Ruxolitinib accelerated the recruitment and killing ability of CD8+ T cells and lowered PD‐L1 expression in cells, thereby suppressing immune escape, which echoed previous studies. Our findings suggested that Amuc_1100 may promote the recruitment and killing ability of CD8+ T cells whilst concurrently suppressing cellular immune escape through inhibiting the JAK/STAT pathway. These effects led to the inhibition of tumour progression in LUAD. However, there is currently no scientific consensus on the quantitative efficacy relationship between AKK and its outer membrane protein Amuc_1100, and different dosages/abundances of AKK may exert different effects, and consistent and reproducible therapeutic outcomes cannot yet be ensured. Therefore, the mechanism of AKK and its outer membrane protein Amuc_1100 in cancer therapy remains to be systematically analysed.

In conclusion, our study illustrated that AKK could accelerate LUAD cells recruiting CD8+ T cells through suppressing the JAK/STAT signalling pathway, enhance the killing ability of CD8+ T cells and inhibit the immune escape of cells. Besides, the main substance of AKK that regulated the immune escape of LUAD cells was the outer membrane protein Amuc_1100. It is important to note that there are some limitations to our study. In this study, the effect of AKK on the immune escape of LUAD cells was only investigated at the cellular level and in nude mice for validating its regulatory role, not in clinical samples. Studies in clinical samples may provide a more direct understanding of the real clinical situation and further validate our observations in cellular and animal experiments. In addition, whilst the nude mouse model has some application in research, it still does not fully reflect the complex immune system of the human body. Therefore, research findings need to be explored in greater depth before they can be translated into clinical practice. Our future endeavours will encompass an extensive evaluation of the long‐term efficacy and safety of Amuc_1100 in cancer treatment, aiming to provide a theoretical basis for its clinical application. To summarize, this study illustrated the crucial role of AKK as a key regulator of intestinal homeostasis, indicating its potential as a promising therapeutic target for LUAD.

AUTHOR CONTRIBUTIONS

Yufen Xu: Conceptualization. Xiaoli Tan: Formal analysis. Qi Yang: Data curation. Zhixian Fang: Writing – original draft. Wenyu Chen: Writing – review and editing.

FUNDING INFORMATION

This work was supported by the Natural Science Foundation of Zhejiang province (no. LQ20H160057); the Key Construction Disciplines of Provincial and Municipal Co construction of Zhejiang (no. 2023‐SSGJ‐002); Scientific Technology Plan Program for Healthcare in Zhejiang Province (nos. 2021RC031, 2022KY377, 2023KY1196 and 2023RC098); Science and technology project of Jiaxing (nos. 2019AY32030, 2020AY30012 and 2021AY30024); National Oncology Clinical Key Speciality (2023‐GJZK‐001) and Jiaxing Key Laboratory of Precision Treatment for Lung Cancer.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

This study has been approved by the Animal Ethics Committee of The Affiliated Hospital of Jiaxing University, approval number: JUMC2023‐046.

CONSENT

All authors consent to submit the manuscript for publication.

Xu, Y. , Tan, X. , Yang, Q. , Fang, Z. & Chen, W. (2024) Akkermansia muciniphila outer membrane protein regulates recruitment of CD8 + T cells in lung adenocarcinoma and through JAK–STAT signalling pathway. Microbial Biotechnology, 17, e14522. Available from: 10.1111/1751-7915.14522

Yufen Xu, Xiaoli Tan and Qi Yang have contributed equally.

DATA AVAILABILITY STATEMENT

The data and materials in the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data and materials in the current study are available from the corresponding author on reasonable request.

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