Toll-Like Receptor 7-Expressed Macrophages Are Involved in the Pathogenesis of Esophageal Achalasia and Esophagogastric Junction Outflow Obstruction

Esophageal achalasia is a typical esophageal motility disorder (EMD). It is defined by the lack of esophageal peristalsis and inadequate relaxation of the lower esophageal sphincter (LES), with symptoms such as dysphagia, regurgitation, chest pain, and weight loss [1]. The pathophysiology of esophageal achalasia reportedly includes decreased or absent ganglion cells in the myenteric plexus and inflammatory cell infiltration into the plexus [2, 3]. Additionally, while previous studies have proposed that viral infections [4‒7], genetic factors [8, 9], and autoimmune diseases [10] are potential causes, the definitive cause remains unclear due to the rarity of the disease.

Viral infections, including measles virus and varicella-zoster virus (VZV), are reportedly associated with esophageal achalasia [4, 5]. However, the limited number of cases described in these reports necessitates caution when drawing conclusions.

Although evaluation of esophageal muscle layer samples is necessary to elucidate the cause of esophageal achalasia, they are difficult to collect during a routine esophagogastroduodenoscopy (EGD) biopsy. Recently, biopsies of the muscle layer have been performed during per-oral endoscopic myotomy (POEM) procedures [11, 12]. Studies have reported pathological changes in the muscles of patients with esophageal achalasia using this technique [6, 11, 12].

Innate immunity is the first defense mechanism against viral and bacterial pathogens invading the human body. Pattern recognition receptors (PRRs) recognize pathogens and induce the expression of interferons and cytokines [13]. Since viral infections have been implicated in the pathogenesis of esophageal achalasia, we hypothesized that innate immunity might be associated with the development and exacerbation of esophageal achalasia. This study aimed to determine the expression of PRRs in the development and exacerbation of esophageal achalasia and esophagogastric junction outflow obstruction (EGJOO) using esophageal muscle specimens collected during POEM procedures.

This was a single-center retrospective study. The study protocol was approved by the Ethics Committee of Hirosaki University (2017-091, 2021-087); this study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki. All patients provided written informed consent to participate.

Patients diagnosed with esophageal achalasia and EGJOO who underwent POEM at our hospital between October 2018 and March 2022, were included in the EMD group. The following data were collected from medical records: age, sex, duration of symptoms, Eckardt score, high-resolution manometry (HRM) diagnosis, integrated relaxation pressure (IRP), esophageal type, and dilation grade on esophagography.

The control group included patients with esophageal squamous cell carcinoma (ESCC) who underwent surgical resection without neoadjuvant chemotherapy and whose tumors did not extend into the esophagogastric junction. The following data were collected from the medical record database: age, sex, and tumor location.

The Eckardt score is a symptom rating for EMD. This score is the sum of the frequency of dysphagia, regurgitation, chest pain, and degree of weight loss [14]. The score for each component ranges from 0 to 3. The higher the score, the more pronounced the symptoms (maximum: 12; minimum: 0).

Each patient with EMD was diagnosed using esophagography, EGD, and HRM based on the Chicago Classification criteria v3.0 [15]. This study employed the Starlet HRM system (Starmedical Ltd, Tokyo, Japan) for diagnosis. IRP was measured as the lowest 4-s cumulative pressure values that occurred during a 10-s post-deglutition time window in the electronically generated e-sleeve signal through the anatomic zone defining the esophagogastric junction [16]. On Starlet, IRP of ≥26 mm Hg was defined as a high IRP value indicating incomplete LES relaxation [17, 18]. The diagnosis of achalasia with normal IRP value was made comprehensively using the typical findings of esophagography as bird-beak appearance with the retention of contrast medium and EGD as the appearance of rosette-like esophageal folds [19] or pinstripe pattern [20]. Esophageal achalasia was further divided into three subtypes based on the type of esophageal contraction: type I (100% failed peristalsis), type II (≥20% pan-esophageal pressurization), and type III (≥20% spastic contraction). EGJOO was diagnosed when the IRP value was high and peristalsis remained normal.

The esophageal type was classified based on the angle of esophageal flexion (α) using a barium esophagogram [21]. The classification is as follows: straight type (St), α ≥ 135°; sigmoid type (Sg), 90° ≤ α < 135°; and advanced sigmoid type (aSg), α < 90°. The esophageal dilation grade is based on the maximum transverse diameter (d) measured. It was classified into the following grades: grade I, d < 3.5 cm; grade II, 3.5 cm ≤ d < 6.0 cm; and grade III, d ≥ 6.0 cm.

POEM was performed on all patients in the EMD group [22, 23]. Biopsies of the esophageal inner circular muscle were performed using biopsy forceps (Radial Jaw 4; Boston Scientific, Natick, MA, USA) during the POEM procedure.

In the control group, samples were removed from the LES of the surgical resection specimens with sufficient margin for the tumor. Subsequently, only the muscle layers were collected.

The muscle layer specimens collected were immediately placed in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was extracted from esophageal muscle layer samples (the EMD and control groups) using the RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany) with a TissueLyser II (QIAGEN), according to the manufacturer’s instructions. Total RNA (200 ng) was reverse-transcribed into complementary DNA (cDNA) using PrimeScript RT Master Mix (TAKARA BIO, Tokyo, Japan). The TaqManGene Expression Assay (Thermo Fisher Scientific) for qPCR was used with the following probes: Toll-like receptor (TLR)2 (Hs02621280_s1), TLR3 (Hs01551078_m1), TLR4 (Hs00152939_m1), TLR7 (Hs01933259_s1), TLR9 (Cj06032976_m1), retinoic acid-inducible gene-I (RIG-I) (Hs01061436_m1), melanoma differentiation-associated protein 5 (MDA5) (Hs00223420_m1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Hs02786624_g1). Reactions were performed using CFX connect (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with Brilliant III Ultra-Fast qPCR Master Mix (Agilent Technologies, LaJolla, CA, USA) under the following conditions: for initial polymerase activity, cycles of 3 min at 95°C, 5 s at 95°C, and 10 s at 60°C were repeated 40 cycles. The results were analyzed using the comparative Ct method, and the expression levels were normalized to the expression level of GAPDH. A sample without cDNA was used as a negative control.

The relationships between TLR7 mRNA expression and the duration of symptoms, Eckardt score, IRP, esophageal type, and dilation grade were investigated. To compare the ratios, the esophageal type was divided into two groups: non-sigmoid (St) and sigmoid (Sg and aSg). Dilation grade was also divided into two groups: maximum transverse diameter <3.5 cm (grade I) and maximum transverse diameter ≥3.5 cm (grades II and III).

The EMD group and control group muscle layers were fixed in formalin and embedded in paraffin before being sliced into 4 μm sections. Immunohistochemical staining was performed using the Ventana Benchmark HX Autostainer (Ventana Medical Systems, Tucson, AZ, USA). Tissue samples were incubated for 32 min at 37°C with anti-TLR7 (1:500, NBP2-24906; Novus Biologicals, Centennial, CO, USA), anti-S-100 (1:1, polyclonal; Nichirei Bioscience, Tokyo, Japan), or anti-CD68 antibodies (1:100, PG-M1; Dako Corp, Tokyo, Japan). An iVIEW Universal DAB Detection Kit (Ventana Medical Systems) was used to detect the bound primary antibodies.

The counts of TLR7-positive mononuclear cells and CD68-positive mononuclear cells per field of view (×400 magnification) were assessed in four separate areas of each specimen. Median counts of TLR7-positive cells and CD68-positive cells in the EMD and control groups were calculated.

Immunofluorescence (IF) staining was performed to determine the expression of CD68 TLR7 double-positive cells. After heat-induced antigen retrieval using an autoclave, the tissue sections were blocked with 5% normal goat serum for 1 h at room temperature (20–25°C). The sections were incubated with mouse monoclonal anti-CD68 (1:250, 14-0688; Thermo Fisher Scientific) and rabbit polyclonal anti-TLR7 (1:250, NBP2-24906; Novus Biologicals) antibodies overnight at 4°C. After washing with phosphate buffer solution, tissue sections were incubated with Alexa Fluor 488 anti-mouse IgG (1:250, A11029; Thermo Fisher Scientific) and Alexa Fluor 594 anti-rabbit IgG (1:250, A11012; Thermo Fisher Scientific) antibodies for 1 h at room temperature. The slides were mounted with ProLong Gold Antifade Reagent (Thermo Fisher Scientific) and visualized using a confocal laser scanning microscopy (C1si; Nikon, Tokyo, Japan).

Statistical analysis was performed using EZR (Jichi Medical University, Shimotsuke, Japan) [24]. The Mann-Whitney U test, Fisher’s exact test, and Spearman’s rank correlation coefficient were used as appropriate. Statistical significance was set at p < 0.05.

Muscle layer specimens were collected from 13 patients who underwent POEM in the EMD group and 5 in the control group. Table 1 presents the clinical background characteristics of the EMD and control groups. In the EMD group, the median (interquartile range [IQR]) age was 63 (54–74) years. Ten male and 3 female patients were included in the analysis. The Chicago Classification of the 13 patients in the EMD group was as follows: 10 patients had type I achalasia, 2 had type II achalasia, and 1 had EGJOO. The median (IQR) duration of symptoms was 8 (6–12) years, Eckardt score was 5 (3–6) points, and IRP was 21.3 (15.5–36.8) mm Hg. The esophageal types were St (n = 6), Sg (n = 6), and aSg (n = 1). The degree of dilation was classified as grade I in three cases, grade II in nine, and grade III in one. In the control group, the median age was 66 years, and 5 male patients were included. The tumor locations in the control group were as follows: 1 patient had a tumor located in the upper thoracic esophagus, 3 patients in the middle thoracic esophagus, and 1 patient in the lower thoracic esophagus.

Figure 1 compares the mRNA expression levels of each PRR examined. The mRNA expression of TLR7 was significantly higher in the EMD group than in the control group. Conversely, mRNA expression levels of TLR2, 3, 4, 9, RIG-I, and MDA5 were not significantly different.

The correlations between TLR7 mRNA expression and duration of symptoms, Eckardt score, and IRP were also examined (shown in Fig. 2). None of these clinical characteristics correlated with TLR7 mRNA expression. Additionally, the correlations between TLR7, esophageal type, and dilation grade were not significant (shown in Fig. 3).

Hematoxylin and eosin and immunohistochemical staining (TLR7, S-100, and CD68) of the muscle layer specimens are shown in Figures 4 and 5. TLR7 and S-100 were positively stained in the myenteric plexus of both the EMD (Fig. 4a–c) and control groups (Fig. 4d–f). This suggests that TLR7 was expressed in Schwann cells.

In some cases, TLR7-positive mononuclear cells were found between the muscle layers. These signals coincided with CD68-positive macrophages. TLR7-positive macrophages were more prevalent in the EMD group (shown in Fig. 5a, b) than in the control group (shown in Fig. 5c, d).

The median (IQR) count of TLR7-positive cells was 6 (4–10)/high-power field (HPF) in the EMD group and 5 (4–7.3)/HPF in the control group. Although a trend toward increased cell counts was observed in the EMD group, the difference was not statistically significant (p = 0.34) (Fig. 6a). Conversely, the median count of CD68-positive cells was 13 (9.5–19.5)/HPF in the EMD group and 4.5 (3.0–7.3)/HPF in the control group, indicating a significant elevation in the EMD group (p < 0.01) (Fig. 6b). IF staining demonstrated that the median (IQR) count of CD68 TLR7 double-positive cells in the EMD group was significantly higher than in the control group (3 [1–4] and 1.5 [0.3–3], respectively) (Fig. 7a, b).

In this study, we found that TLR7 mRNA expression was upregulated in the muscle layers of patients with the EMD group compared with that of the control group. Histological staining revealed that TLR7 expression was consistent with macrophages between the muscle layers. Additionally, CD68 TLR7 double-positive cells significantly increased in the EMD group muscle layers. These results suggest that TLR7 in macrophages might be involved in the innate immune response underlying esophageal achalasia pathogenesis.

TLR1-10, RIG-I, and MDA5 are PRRs that are expressed in various human cells. Among these PRRs, TLR2, 3, 7, 9, RIG-I, and MDA5 recognize viral infection [25‒27]. In this study, the analysis of mRNA expression showed that TLR7 mRNA expression was significantly higher in the EMD group than in the control group. TLR7 is reportedly expressed predominantly in plasmacytoid dendritic cells and macrophages [13, 28]. TLR7 is localized within intracellular endosomes and recognizes virus-derived single-stranded RNA (ssRNA). Specifically, TLR7 reportedly recognizes coxsackievirus B, influenza virus, vesicular stomatitis virus, human immunodeficiency virus-1, hepatitis B virus, and hepatitis C virus [29‒34]. TLR7 signaling, recognizing ssRNA, induces type I interferon production via myeloid differentiation factor 88 (MyD88) and nuclear factor-kappa B (NF-κB) [29, 35‒37]. One previous study has shown that TLR7 is expressed in Schwann cells obtained from the sciatic nerve in a study using rats. Moreover, NF-κB is activated by stimulating Schwann cells with R848 (TLR7/8 ligand) [38].

Palmieri et al. [39] investigated the miRNAs in the esophageal muscle layers collected during surgical myotomy in 11 patients with achalasia and esophageal muscle layers from 5 patients following gastric cancer surgery. They reported a significant elevation of miR-122 and -133a in esophageal achalasia. In another study using mouse ischemic myocardium, miR-122 and -133a were suggested to be associated with the cytokine response downstream of TLR7 and MyD88 [40]. Based on these reports, a possibility exists that TLR7 is involved in the pathogenesis of achalasia, which is consistent with our research findings showing elevated TLR7 mRNA expression in the EMD group. Therefore, the development of achalasia might involve an immune response mediated by TLR7, which recognizes certain viruses.

Immunohistochemical staining showed that TLR7 positivity in Schwann cells in both groups had no discernible differences. This suggests that TLR7 is constitutively expressed in Schwann cells and might be involved in the antiviral response regardless of the pathogenesis of esophageal achalasia. Conversely, the number of TLR7-positive macrophages was higher in the EMD group compared with that of the control group. This might be attributed to the significant increase in TLR7 mRNA expression in the EMD group.

In this study, no correlation was found between TLR7 mRNA expression and the duration of symptoms. Ikebuchi et al. [7] reported a significant elevation of herpes simplex virus-1 (HSV-1)-derived miRNA in the achalasia group compared with that of the control group, which did not correlate with the duration of symptoms. Additionally, another study demonstrated an increase in M1 macrophages, which are known to promote inflammation, in the achalasia group; however, no correlation was observed for the duration of symptoms [41], which is consistent with the findings of our study. The innate immune response to certain viruses via TLR7 in macrophages is presumed to occur early during the onset of esophageal achalasia. However, as POEM is typically performed several years after disease onset, assessing the immune response during the initial stages of the disease is difficult. The absence of a correlation between TLR7 mRNA expression and the duration of symptoms found in our study suggests that an aberrant immune response during the initial stage of disease onset might have persisted until the time of treatment. Novel therapeutic targets for this persistent response might be found in the future with additional research.

While no significant difference was observed, a tendency for increased expression of TLR7 in the group with sigmoid type and severe esophageal dilation was observed. The accumulation of esophageal residues might lead to bacterial fermentation and stasis esophagitis, potentially inducing the expression of TLR7. However, prolonged disease duration generally leads to esophageal bending, increased dilation, and easier accumulation of residues in the esophagus. Since no correlation was found between TLR7 and the duration of symptoms in this study, the involvement of bacterial fermentation and stasis esophagitis by esophageal residues in TLR7 expression is limited.

Several studies have focused on viral infections as a cause of esophageal achalasia. A previous study demonstrated upregulation of hsv-1-miR-H1 and -H18, derived from HSV-1, in the esophageal muscle layer collected during POEM procedures, as compared to that of control specimens [7]. Jones et al. [4] evaluated the serum antibody titers of nine viruses, including measles viruses, in 18 patients with achalasia and 12 normal controls; overall, the antibody-positive rate for measles virus was significantly higher in patients with esophageal achalasia. Robertson et al. [5] collected muscle layers from patients with esophageal achalasia during Heller surgery and evaluated VZV levels using fluorescence in situ hybridization. They reported that VZV was detected in 3/9 cases. Based on these findings, we hypothesized that TLR2, TLR9, RIG-I, and MDA5 might be involved in the development of esophageal achalasia and EGJOO as they recognize these viruses [25, 42‒44]. The mRNA expression of TLR2 and TLR4 was lower in the EMD group than in the control group (Fig. 1). A correlation between TLR2 and duration of symptoms was examined, revealing a significant negative correlation (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000540693). In addition, a negative correlation was also observed between TLR4 and the duration of symptoms; however, it was not significant (online suppl. Fig. 2). Chronic inflammation due to stasis esophagitis associated with the progression of achalasia may have affected the decreased expression of these mRNA.

This study had some limitations. First, this was a single-center, retrospective study. Second, the relatively small volume of specimens obtained using biopsy forceps was small. Therefore, whether these specimens reflected the entire esophageal muscle layer in esophageal achalasia and EGJOO cases is unclear. Furthermore, due to the small amount of total RNA extracted, examining the expression of cytokines and chemokines by PCR was not possible. Additionally, the comparison of Schwann cell density or the number of nerve ganglia between the two groups might not be appropriate because the control group used whole-layer specimens including the inner circular muscle and outer longitudinal muscle, while the EMD group used only the inner circular muscle. Third, most patients with esophageal achalasia and EGJOO in this study underwent POEM from several years to more than a decade after the onset of these disorders. Therefore, the initial state of esophageal achalasia has not yet been assessed. Fourth, not all cases showed TLR7-positive macrophages between the muscle layers. Fifth, the muscle layer of the non-neoplastic part of the esophageal squamous cell carcinoma was used as a control. This might be different from that observed in the healthy subjects who would be the optimal control. However, harvesting the muscle layers from healthy individuals was technically and ethically challenging.

At the outset of this study, attempts to manually homogenize muscle layer specimens for total RNA extraction were unsuccessful. Subsequently, mechanical homogenization enabled the successful extraction of total RNA. To ensure procedural safety, the amount of muscle tissue that can be collected must be small. Therefore, the number of patients in which sufficient RNA can be collected for analysis is limited. Few studies have focused on the mRNA expression of PRRs by extracting total RNA from the esophageal muscle layers. This is an important result of our study, despite some limitations.

In conclusion, we revealed the association between esophageal achalasia and PRRs using esophageal muscle layer specimens collected during POEM procedures. Increased TLR7 expression in macrophages between muscle layers might be involved in the pathogenesis of esophageal achalasia. Furthermore, the regulation of cellular and immune responses may serve as a new therapeutic target.

We would like to thank all the members of the Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, for assisting with the immunostaining assay. We also wish to thank Editage (www.editage.jp) for English language editing.

This study protocol was reviewed and approved by the Ethics Committee of Hirosaki University, Approval Nos. 2017-091 and 2021-087. Written informed consent was obtained from participants to participate in the study.

The authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

Study conception and design: Tetsuya Tatsuta. Data curation and acquisition: Masatoshi Kaizuka, Tetsuya Tatsuta, Tetsuyuki Tateda, Yohei Sawada, Shinji Ota, Shiro Hayamizu, Keisuke Hasui, Hidezumi Kikuchi, Hiroto Hiraga, Daisuke Chinda, Takahiro Muroya, mRNA analysis: Masatoshi Kaizuka, Tetsuya Tatsuta, Shogo Kawaguchi, Shukuko Yoshida, Shinji Ota. Histological analysis: Tadashi Yoshizawa and Hiroshi Kijima. Manuscript preparation: Masatoshi Kaizuka, Tetsuya Tatsuta, Shogo Kawaguchi. Writing – review and editing: Tatsuya Mikami, Kenichi Hakamada and Shinsaku Fukuda. Study supervision: Hirotake Sakuraba. Final approval of the article: all authors.

All date are presented in this paper. Further inquiries can be directed to the corresponding author.