Idiopathic achalasia: unraveling the etiology and pathogenesis of an inflammatory disease

Abstract

Idiopathic achalasia is a rare esophageal motility disorder caused by the loss of enteric neurons leading to impaired relaxation of the lower esophageal sphincter and absent peristalsis of the esophageal body. Despite being described over three centuries ago, the precise etiology and molecular pathogenesis of idiopathic achalasia remain unclear. Recent studies indicate that an initial injury to the esophagus, potentially caused by viral infection, may trigger inflammation in genetically predisposed individuals. Ultimately, chronic inflammation causes the destruction of inhibitory myenteric neurons, resulting in clinical symptoms of achalasia-like dysphagia, regurgitation, chest pain, and weight loss. Although extensive research has been conducted on the etiology of achalasia, the majority of these studies have been limited to exploration of the infection and immune factors within the context of genetic susceptibility. In this review, we aim to provide a detailed understanding of the etiology of achalasia. In addition to the genetic factors, viral infection, and autoimmunity, we additionally propose that psychological factors, gut microbiota, and the gut–brain axis play a role in the pathogenesis of achalasia.

Introduction

Idiopathic achalasia is a primary esophageal motility disorder of indeterminate etiology, manifesting as esophageal aperistalsis and failure of the lower esophageal sphincter (LES) to relax appropriately during swallowing, as demonstrated through manometric assessments. This rare disease exhibits an annual incidence rate of ∼1–5 cases per 100,000 people and a prevalence of 7–32 cases per 100,000 people globally [1–4]. The reported incidence varies by region; for example, in Chicago, USA, when the most advanced technology and diagnostic standards are used, it ranges from 1.35 to 4.60 per 100,000 [5], whereas, in South Australia, it is ∼2.3–2.8 per 100,000 [6]. Clinical symptoms encompass dysphagia for solids and liquids, regurgitation, chest pain, and weight loss [7, 8]. With high-resolution manometry, which is the gold standard for diagnosing it, achalasia is diagnosed as an integrated relaxation pressure of ≥15 mmHg lasting for 4 seconds and it can be subclassified into three types according to the motility patterns of the esophageal body: type I is characterized by 100% absent body peristalsis without pressurization, type II by ≥20% pan-esophageal pressurization, and type III by abnormal body peristalsis with two or more premature contractions [9].

Although achalasia has been documented for over three centuries, its etiology and pathogenesis remain enigmatic [10]. Extensive research has proved an unidentified degenerative process [8, 11, 12] that results in the loss of ganglion cells in the esophageal myenteric plexus, accompanied by the infiltration of immune cells [11]. Subsequently, critical inhibitory neurotransmitters such as vasoactive intestinal peptide (VIP) and nitric oxide (NO) are lost [13, 14]. The loss of these neurotransmitters disrupts the normal contraction of the LES smooth muscle, and the immune-cell infiltration suggests that autoimmune and inflammatory responses play a key role in the etiology of achalasia (Fig. 1). In the early phase, esophageal inhibitory nerve degeneration leads to unopposed activity of excitatory neurotransmitters such as acetylcholine, causing high-amplitude non-peristaltic contractions (vigorous achalasia). Over time, the gradual loss of cholinergic neurons results in esophageal dilation and low-amplitude synchronous contractions (classic achalasia). Substantial reports have shown that the etiology of achalasia is multifactorial, involving the enteric neuron inflammation triggered by viral infection and poison infection such as aflatoxin [15] in genetically susceptible individuals (Fig. 2).

Figure 1.

The entire process of the pathogenesis of achalasia. Among the genetically susceptible population with genetic defects, viral infection induces a local inflammatory response in the LES. Various cells and inflammatory factors gather in the inter-muscular plexus, resulting in the loss of inhibitory neurotransmitters VIP and NO. Eventually, this leads to persistent abnormal contractions of the LES and the appearance of achalasia symptoms. ALADIN = the product of the AAAS gene, which means achalasia–addisonianism–alacrima syndrome, VIPR1 = vasoactive intestinal peptide receptor 1, PTPN22 = protein tyrosine phosphatase non-receptor 22 gene, IL-23R = interleukin-23 receptor, MV = measles virus, HSV = herpes simplex virus, CMV = cytomegalovirus, CD4 T cell = cluster of differentiation 4-positive T lymphocytes.

Figure 2.

Discovery of the etiology of achalasia of the cardia, including genetic susceptibility, infection, autoimmunity, psychological factors, gut microbiota, and gut–brain axis. HPV = human papillomavirus, MV = measles virus.

Genetic factors

Genetic factors are hypothesized to significantly influence the etiology and advancement of achalasia. Evidence from familial occurrences and sibling cases [16] suggests a potential heritable nature of achalasia. The increased prevalence among African Americans and Hispanics further indicates a genetic contribution, possibly related to racial factors [17]. Additionally, statistical associations between achalasia and specific defined genetic syndromes have been documented, further supporting the involvement of genetic factors in the pathogenesis of this disease. For example, many esophageal motility disorders were reported as closely related to Down’s syndrome [18]. Besides, familial visceral neuropathy [19], Alport syndrome [20], and other genetic diseases were also reported to be related to achalasia in recent studies.

This review summarizes novel gene mutations associated with achalasia. Extensive genetic sequencing studies have identified various genetic mutations in patients with achalasia. Our team successfully validated three rare variants (CREB5, GenBank: NC_000007.13: g.28848865G>T; ESYT3, GenBank: NC_000003.11: g.138183253C>T; and LPIN1, GenBank: NC_000002.11: g.11925128A>G) that heighten the risk of developing achalasia [21]. Other variants, such as those in CRLF1 [22], SAMD9 [23], and FOXP1, have been implicated in haploinsufficiency [24]. Additionally, biallelic CAV1 null variants [25] and the prion gene PRNP Y162X truncation mutation [26] have also been identified in achalasia patients. Besides, a genotyping study revealed that the single nucleotide polymorphism (SNP) rs1799724, located between the lymphotoxin-α and tumor necrosis factor-α (TNF-α) genes, is significantly associated with achalasia [27]. Additionally, three other SNPs (located in MYO5B, ADRB2, and IL13 genes) were found to be associated with achalasia in the meta-analysis. These results indicate that many gene locus mutations related to inflammatory factors play an important role in the susceptibility factors of achalasia of the cardia, suggesting the significance of genetic factors in etiology and their close relationship with the inflammatory response.

Although the co-occurrence of genetic diseases with achalasia is well documented, this evidence alone is insufficient to establish a genetic etiology for the disease. The following discussion will elaborate on the role of genetic factors in the pathogenesis of achalasia. Genetic disorders may predispose individuals to viral infections and subsequent neurodegenerative changes, which increase the possibility of developing this disease. Other factors such as viral infections, autoimmunity, psychological factors, gut microbiota, and the gut–brain axis can also play a role in the development of achalasia.

Genes mediating autoimmune response processes

Human leukocyte antigen association

The human leukocyte antigen (HLA) Class II antigen is located on chromosome 6p21 and spans ∼700 kb. This region encompasses >30 genes, including HLA-DP, HLA-DQ, and HLA-DR [28], which have been implicated in numerous autoimmune disorders. Notably, the major histocompatibility complex class II (MHC II) encoded by HLA is not directly associated with cytotoxic T cells (CD8+ T cells) because CD8+ T cells primarily recognize and kill infected or abnormal host cells through the MHC I molecule. Thus, the function of MHC II is closely related to humoral immunity in the adaptive immune system rather than a cytotoxic response. Recently, the research on HLA class II highlighted that aberrations in the autoimmune response were pivotal in the pathogenesis of achalasia [29]. Specifically, T-cell lymphocytes, capable of recognizing specific HLA class II antigens, constituted the primary cellular component within the infiltrates of the esophageal myenteric plexus in patients with achalasia [30].

Among the major HLA gene families mentioned, HLA-DQB1 has received the most attention. A genetic study [31] precisely located a significant MHC association signal linked to an eight-residue insertion in HLA-DQB1, encoded by HLA-DQB1 05:03 and HLA-DQB1 06:01. This eight-residue insertion conferred the most decisive risk factor for achalasia. Further analysis [32] revealed that this insertion displayed a geospatial north–south gradient in frequency. A subsequent genotype–phenotype analysis [33] showed that the HLA-DQB1 insertion frequency varied among achalasia subtypes, providing insights into its genetic etiology. Notably, the insertion was most frequently observed in type I, indicating that immune-mediated mechanisms triggered by this genetic variant may play a more significant role in the pathogenesis of this subtype.

Besides HLA-DQ, HLA-DR alleles were also reported to be associated with achalasia. A recent study reported significant elevations in the frequencies of alleles DRB1*14:54 [34], as well as the extended haplotypes DRB1*14:54 and DRB1*11:01. Our team performed whole-exome sequencing (WES) on 100 achalasia patients and 313 control subjects, revealing a reproducible association between the common missense variant of HLA-DPB1 (rs1126511) located at 6p21.32 and an increased risk of achalasia [21]. Additionally, by analysing the WES data, we inferred the HLA loci and successfully determined the genotype of six HLA genes. Our findings identified two HLA-DPB1 loci (DPB1*21:01, DPB1*17:01) and one HLA-DRB1 locus (MIM: 142857; DRB1*07:01) as contributors to the risk of achalasia.

In achalasia, B cells produce autoantibodies against antigens derived from esophageal ganglion cells—a process facilitated by HLA-II variants presenting these antigens to T helper cells. These autoantibodies contribute to disease pathology through several mechanisms. First, they activate the complement system, leading to the formation of membrane attack complexes that lyse ganglion cells. Second, autoantibodies mediate antibody-dependent cellular cytotoxicity, in which natural killer cells destroy ganglion cells bound by antibodies. Autoantibodies can interfere with ganglion-cell functions, disrupting neurotransmitter release, such as NO, which is crucial for LES relaxation. At present, the genes related to B-cell-mediated inflammation have not been discovered and more research is urgently needed.

It is worth noting that the vast majority of the above-mentioned genes related to autoimmunity remain at the sequencing stage, without conducting validation through basic experiments and animal experiments. Therefore, the validation of downstream gene pathways of HLA has become a current research hotspot.

Protein tyrosine phosphatase non-receptor 22 gene polymorphism

The PTPN22 gene [35], located on chromosome 1p13.3-p13, encodes the protein tyrosine phosphatase non-receptor 22—a key regulator of immune function. It encodes the lymphoid-specific phosphatase that downregulates T-cell activation [36]. PTPN22 may contribute to disease development by dysregulating T-cell activation and autoimmunity. Specifically, it dephosphorylates key signaling molecules such as lymphocyte-specific protein tyrosine kinase and tyrosine-protein kinase Fyn, thereby suppressing T-cell activation. Mutations such as the R620W polymorphism can alter PTPN22 function and are associated with increased risk for autoimmune responses, potentially leading to the immune-mediated destruction of esophageal ganglion cells seen in achalasia [37, 38]. Interestingly, a case–control study by Santiago et al. [39] involving 231 unrelated Spanish patients of White ethnicity with achalasia and 554 healthy control subjects found that the frequency of the 1858 T allele was higher in female achalasia patients than in healthy controls. This result revealed a gender-specific susceptibility, with a potentially more pronounced effect in women. This difference may be attributed to the inherent disparities in basic immune responses between the genders in which alterations in T-cell signaling primarily impact women.

Genes mediating esophageal motor function

NO synthase polymorphism

NO serves as a crucial inhibitory neurotransmitter within the esophageal myenteric plexus [40–42], playing a pivotal role in the LES relaxation [43]. NO synthase (NOS) catalyses the production of NO from L-arginin. In 1993, Mearin et al. [44] pioneered in reporting the absence of NOS activity in LES tissue samples from patients with achalasia. Subsequent research has confirmed the significance of NO as the primary inhibitory neurotransmitter [41, 44]. Three distinct forms of NOS were identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). These forms are encoded by three unique genes: NOS1 (encoding nNOS), NOS2 (encoding iNOS), and NOS3 (encoding eNOS), which are situated on chromosomes 12q24, 17q11, and 7q36 [45]. As achalasia is a neurodegenerative disease, there is no doubt that nNOS is most closely associated with this condition.

In patients with achalasia, studies have postulated that the loss of NO-secreting esophageal myenteric plexus neurons results in an imbalance between excitatory and inhibitory neural signaling, ultimately contributing to alterations in esophageal manometry findings. While both inducible iNOS and nNOS mRNA have been detected in both achalasia and control tissues, immunohistochemical analysis has revealed positive staining for nNOS alone [46].

Numerous case–control studies on this aspect of gene phenotypes have shown that nNOS29TT genotypes were more prevalent among achalasia patients compared with the control group [47]. Similar experiments were also reflected in the functional polymorphism (CCTTT) of the NOS2 promoter [48] and truncating mutations in the NOS1 gene [49]. These genetic phenotypic mutations have been previously linked to various autoimmune disorders and are critical in regulating NOS gene expression, indirectly highlighting the role of autoimmune factors in achalasia.

In addition to the intragenic variations within NOS genes, the signaling pathway modulated by NOS also represents a significant area of scientific inquiry. The NO–cGMP–PKG signaling pathway has been implicated in the pathogenesis of achalasia [50]. NO activates soluble guanylate cyclase in vascular smooth muscle cells, promoting cyclic guanosine monophosphate (cGMP) synthesis. This process activates cGMP-dependent protein kinases (cGKs) that regulate ion channels and the actions of cGMP. cGKs inhibit Ca2⁺ leakage from the endoplasmic reticulum, causing smooth muscle relaxation. By targeting the cyclic GMP-dependent protein kinase (PRKG1) gene, down-expression of miR-200c-3p has been proved to contribute to achalasia [50]. Another target of this pathway—soluble guanylate cyclase, the key receptor for NO—is also affected by homozygous mutations including GUCY1A3 [45, 51] and the murine retrovirus integration site 1 (MRVI1) gene [52], which encode the inositol trisphosphate receptor 1 (IP3R1)-associated cGMP kinase substrate. This result suggested that MRVI1 mutations should be evaluated as a potential underlying cause of the disease for individuals who exhibit an early onset of achalasia without any accompanying symptoms.

Investigations have demonstrated a correlation between a diminished network of interstitial cells of Cajal (ICCs) within the esophageal wall and decreased NOS expression in patients diagnosed with achalasia [41, 53]. ICCs occupy a pivotal position in esophageal inhibitory neurotransmission, as they participate in the biosynthesis and liberation of NO [54]. Notably, mutations in the stem cell factor receptor gene [tyrosine kinase receptor (KIT)] have been implicated in gastrointestinal motility disorders in murine models, primarily attributed to the depletion of ICCs [55, 56]. The observation of a substantial reduction in c-KIT expression and overexpression of the inositol polyphosphate-4-phosphatase, type II B gene in achalasia patients suggested novel signaling pathways involved in primary achalasia [57].

VIP receptor-1 gene polymorphism

VIP is a small neuropeptide that functions as a neurotransmitter with notable anti-inflammatory properties [58, 59]. It is found in the myenteric plexus and plays a critical role in modulating the relaxation of the distal esophageal wall and LES. VIP receptors are encoded by the VIP receptor 1 (VIPR1) gene, located on chromosome 3p22 [60]. VIPR1 belongs to the secretin receptor family—a group of G-protein-coupled receptors expressed by immune cells and myenteric neurons [61]. Indeed, it has been reported that sustained inflammation can disrupt VIPR1 signaling [42, 62–64], thereby altering the regulatory influence of VIP on myenteric neurons. This dysregulation can progress to ganglion-cell loss and nerve-fiber fibrosis, and these processes are closely linked to the pathogenesis of achalasia. A study conducted by Paladini revealed significant variations in the allele, genotype, and phenotype distribution of SNP rs437876 within intron 4 among patients with achalasia [65]. These SNPs were subsequently chosen and evaluated for their prevalence among 104 individuals diagnosed with achalasia and a group of 300 randomly chosen controls. It is noteworthy that the association between VIPR1 and idiopathic achalasia, as reported in this study, remains valid among patients who experience a later onset of the disease.

Allgrove syndrome

Triple A (Allgrove) syndrome is a rare autosomal recessive condition that is distinctively characterized by a triad of manifestations: primary adrenal insufficiency stemming from adrenocorticotropic hormone resistance, achalasia, and alacrima [66]. Given its strong genetic factor, it has emerged as a new research hotspot. In addition, the increasing number of familial clustering cases also warrants attention [67, 68]. The similarity of these instances is attributable to alterations in the AAAS gene situated on chromosome 12q13, which encodes for the protein involved in alacrima–achalasia–adrenal-insufficiency neurologic disorders [69]. Nevertheless, clinical manifestations and genetic screening procedures can be intricate due to the variability in symptomology and the inability to detect mutations in all clinically diagnosed individuals. For instance, ∼30% of patients do not carry mutations in the AAAS gene [70]. Therefore, the current research focuses on genetic mutations beyond the AAAS gene. The mutations that have been discovered include a homozygous splice mutation in TRAPPC11 [70] and biallelic mutations in the GMPPA gene [71]. Further genetic sequencing studies on such mutations are being conducted widely.

It is worth noting that, although etiological research on the genes of achalasia is extensive at present and a large number of basic experiments have verified the importance of genes, the biggest challenge in current research lies in constructing appropriate animal models. The vast majority of basic studies focus on phenotypic verification after overexpressing the target gene at the baseline level rather than modeling achalasia mice and then knocking down the targeted gene to discover a decline in phenotypic characteristics.

The current animal models can be classified into three categories. In the first, a chemical neuroinhibition method is used, such as applying a benzalkonium chloride coating on the LES to stably reproduce the high tension and mega esophagus caused by the loss of inhibitory neurons. This method is straightforward and exhibits good repeatability. However, the benzalkonium chloride-induced method can only simulate a phenotype similar to achalasia, as it is fundamentally different from the genetic susceptibility-inflammation and immunity-induced evolutionary process of neural damage in terms of etiology [72, 73]. The second method involves genetic-pathway models such as nNOS deletion and the specific knockout of NO-sensitive guanylyl cyclase (NO-GC) in smooth muscle or ICC cells. This mechanism confirms that the NO-GC–cGMP-ICC pathway is crucial for LES relaxation, making it suitable for target and pharmacological intervention validation [74, 75]. Third, infection/immune models of herpes simplex virus-1 (HSV-1 persistent infection, T. cruzi/Chagas) can reproduce neuroinflammatory damage and mega esophagus, which aligns with the immune trigger-susceptible host etiological hypothesis. However, these methods can only induce the achalasia phenotype under specific conditions of viral infection or autoimmunity, and they are unable to produce stable modeling under normal circumstances [76]. Based on the results of previous sequencing, our team is currently constructing several animal models of gene-edited mice and some of them have developed phenotypes. However, more animal-model construction and validation need to be explored in the future.

Viral infection

In the past decades, several case–control studies found that the serum antibody titers of the measles virus [77], varicella-zoster virus (VZV) [78, 79], and herpes simplex virus type 1 [80] of achalasia patients were significantly higher than those of healthy controls. This suggests the potential involvement of viral infection as a contributing factor in the disease. Viral infections trigger enteric neuron inflammation of the body, so the impact of viral infections on achalasia may be related to the impaired neurons. However, no definitive causal link has been established between viral infection and achalasia, yet the possibility of viral infection serving as a contributory factor cannot be completely excluded. There are many case reports of infectious diseases such as Guillain–Barré syndrome, poliomyelitis [81, 82], and VZV [78]; these cases share a similar virus infection and autoimmune mechanism with achalasia. Moreover, in Chagas’ disease [83], caused by an infection with Trypanosoma cruzi [84], impaired motor function similar to that of the LES in achalasia is also found [85, 86]. Reports have indicated that viruses may induce comparable motor abnormalities in the avian esophagus. Kistler et al. discovered Borna disease virus associated with proventricular dilatation disease in birds, which infected neurons via host T-cell-mediated immune responses to viral antigens displayed on cells [87, 88]. These findings provide evidence that viruses have the potential to elicit pathophysiological processes resembling those observed in achalasia, thereby sparking renewed scientific interest in identifying infectious agents implicated in achalasia.

Herpes virus family

The herpes virus family, which mainly consists of HSV, Epstein–Barr virus, VZV, and cytomegalovirus, was identified in young, healthy persons without immune diseases [89]. This widespread susceptibility indicates that the general population is frequently exposed to these viruses. It is notable that the surface layer of the LES is squamous epithelium. The herpes virus family, as neurotropic viruses, prefer to infect the squamous epithelium [89, 90]. Thus, it is conceivable that infection with these viruses could result in esophageal ganglion-cell destruction, potentially leading to achalasia [91]. A cross-sectional study [11] revealed the association between the subtypes of achalasia and the autoimmune response caused by HSV-1, indicating a link between HSV-1 infection and the development of achalasia.

However, achalasia does not always occur in individuals with viral infections [91]. It is suggested that certain immune-system-related genetic abnormalities may increase predisposition to this disease. This hypothesis has prompted researchers to investigate the mechanism of the virus acting on esophageal neuron cells. Boeckxstaens et al. [92] suggested that, during latent HSV-1 infection, the virus remains dormant in the neurons of the LES and esophagus, leading to a constant immune response. This constant immune activation can potentially trigger neuronal damage in genetically predisposed individuals [93]. A subsequent study [94] implicated that the oligoclonal lymphocytic infiltration observed within the LES of patients with achalasia could potentially serve as a biomarker of an immune-inflammatory response elicited by HSV-1 antigens.

Recently, Japanese scholars investigated the expression of E3 ubiquitin-protein ligase component n-recognition 1 and autophagy-related 16-like 1 in biopsy samples from the LES muscle during per-oral endoscopic myotomy (POEM) for achalasia as potential targets of viral microRNAs [95]. The results showed that the levels of autophagy-related 16-like 1—a target of HSV1-miRH1—were reduced in the LES muscle of achalasia patients, indicating that its reduction could be related to the mechanism of achalasia. It is noteworthy that not all individuals infected with HSV-1 develop achalasia [96]. This observation has led to the hypothesis that most patients might have effectively cleared off the virus or, alternatively, there might be potential sampling errors involved in the detection process, so further studies need to be done to demonstrate the direct evidence of the virus in esophageal tissue.

In addition to HSV, VZV also plays a significant role in the etiology of achalasia [97]. The researchers confirmed the hypothesis that reactivation of latent VZV in esophageal neurons may lead to chronic infection, impairing esophageal motility and LES function. The findings suggest a possible etiological role of VZV in achalasia, opening avenues for further research into the pathogenesis of this disorder.

Severe acute respiratory syndrome coronavirus 2

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [98]. Since its pandemic in 2019, more and more gastrointestinal motility disorders related to it have been reported [99, 100], including achalasia [101–104]. 4A syndrome patients with suspected symptoms of COVID-19 were first reported by Azmoodeh et al. [105]. Recently, studies have proposed that the gastrointestinal tract is highly susceptible to infection by SARS-CoV-2 due to the presence of angiotensin-converting enzyme type II receptors expressed in ileal and esophageal epithelial cells [102, 106].

A central debate in the field revolves around whether or not SARS-CoV-2 is related to the pathogenesis of achalasia. Some scholars have designed experiments to verify this and discovered the inflammatory response mechanism involved. It turned out that coronavirus was found in six of seven achalasia–COVID-19 patients compared with undetectable ones in the achalasia patient and controls, indicating that COVID-19 could affect the myenteric plexus [102]. Related inflammatory cytokines such as Th22, Th17, and Th1, and regulatory plasmacytoid dendritic cells were elevated in both achalasia and achalasia–COVID-19 patients. Similarly, inflammatory markers NOD-like receptor family pyrin domain-containing 3 and TNF were found to be related to achalasia in another group of infected patients [107].

SARS-CoV-2 also has psychological effects on achalasia patients. A recent study revealed that the imposition of COVID-19 lockdown measures exerted an influence on the psychological health and upper gastrointestinal manifestations of individuals suffering from achalasia [108]. Telemedicine could serve as a viable strategy for follow-up care and monitoring, which may explain the huge decrease in POEM and high-resolution manometry activity in Japan [109].

However, not all scholars support this idea. Mohammed et al. assumed that the pathogenesis of achalasia might not involve direct viruses, but rather autoimmune reactions [110]. Achalasia is caused by the immune response of individuals with specific mutations in the HLA-DQ region to viral infections. SARS-CoV-2 may induce autoantibodies through molecular mimicry, leading to various clinical outcomes. In conclusion, extensive studies will be instrumental in elucidating the potential etiology of SARS-CoV-2 as a triggering agent for achalasia. As the COVID-19 pandemic gradually subsides, when hospitals admit patients with achalasia in the future, they need to pay more attention to the correlation between the virus and the disease progression.

Other viruses

In addition, some other viruses, such as measles virus [77], human immunodeficiency virus [111], hepatitis C virus [112], and human papillomavirus [113], were also reported to be related to achalasia. Certain studies have also postulated that immunodeficiency virus may directly contribute to the development of achalasia [114]. The mumps virus, which belongs to the Paramyxoviridae family, has, for the first time, recently been reported to be associated with achalasia [115]. A controversial association between these viruses and achalasia has been documented. In an immunohistochemical study of the myenteric plexus in achalasia [116], all patients were completely negative for the presence of both the herpes simplex virus and the human papillomavirus. Recently, Moradi et al. were unable to establish a substantial link between achalasia and various types of viruses among the subjects examined [96]. Therefore, researchers have not reached a consensus on whether viruses can be a decisive pathogenic factor.

As an important pathogenic factor of achalasia, the virus has been supported by a large amount of clinical data, serological studies, and epidemiological data. However, the corresponding basic experimental work is still far from sufficient, especially animal experiments. Considering that the SARS-CoV-2 was newly discovered in recent years, a large patient cohort and clinical sample bank need to be established. At present, the key role of viral factors in the diagnosis and treatment of achalasia lies in risk prevention, as it is more like the initiating factor in a complete pathogenic chain (Fig. 1).

Inflammation and autoimmunity

Under specific genetic and environmental conditions, chronic viral infection may initiate an abnormal immune response, thereby promoting the inflammatory process, involving unbalanced inflammatory cells, cytokines, chemokines, complements, and autoantibodies. This process will subsequently cause the loss of enteric myenteric neurons. Accumulating evidence has indicated that immune-mediated inflammation is a crucial pathogenesis of inhibitory neuron degeneration in the LES. The process of the inflammatory response is highly complex. The relevant reaction mediators are shown in Fig. 3.

Figure 3.

Specific details of inflammation and autoimmune responses, including various immune cells, inflammatory factors, and autoantibodies. C1r = complement component 1r, C1q = complement component 1q, anti-M2R = anti-muscarinic acetylcholine receptor m2 antibody, PMN = polymorphonuclear.

Inflammatory cells

Inflammatory cells, also known as immune cells or leukocytes, play a crucial role in the body’s inflammatory response. This response is a complex biological process that involves the body’s attempt to eliminate or neutralize harmful stimuli, such as pathogens (bacteria, viruses, and fungi), damaged cells, or irritants. They include lymphocytes, mast cells, granulocytes (eosinophils, basophils, neutrophils), monocytes, and macrophages.

Eosinophils

Recent studies showed that achalasia was associated with infiltration of activated eosinophils in the esophageal muscle [117–119] and esophageal motility disorder of eosinophilic esophagitis (EoE) [120, 121]. Activated eosinophils induce tissue damage through the release of cytotoxic eosinophil-specific proteins, including major basic proteins, eosinophil-derived neurotoxins, and eosinophilic cationic protein [122]. The current debate focuses on whether eosinophilia is a consequence of the esophageal stasis of achalasia or an emerging cause of achalasia [123]. Esophagus movement issues can be ameliorated with steroids, which also lower eosinophil levels, suggesting that these cells might be causing the temporary motility disturbances [124]. In order to investigate whether eosinophils only cause transient esophageal motility disorders, researchers used mouse models to demonstrate the impact of eosinophil infiltration on esophageal motor dysfunction in the lower esophagus, accompanied by the downregulation of SOX-10-IR cells [125]. Recently, Reddy et al. proposed an alternative hypothesis that achalasia might sometimes be allergy-driven [118], as they found a significant association between achalasia and EoE as well as other allergic conditions, which may create a microenvironment conducive to tumor growth such as esophageal cancer. These findings suggested that achalasia might result from EoE and vice versa. However, current studies are all limited to verifying the symptom correlation between achalasia and EoE at the eosinophilic cell level. The specific molecular mechanism has still to be discovered, emphasizing the need for larger multicenter studies to further investigate this complex interaction.

Mast cells

Mast cells contain histamine and heparin. After being activated, they can release granules containing peptides, cytokines, enzymes, and growth factors into the surrounding tissue. These molecules modulate immune and neuronal cells, influencing acquired immune responses. When mast cells accumulate in the esophagus, their myoactive, pro-inflammatory, and cytotoxic products have the potential to induce achalasia-like motility disorders and neuronal degeneration [126]. There are reports identifying mast cells presenting in the esophageal musculature of patients with achalasia [127], even in EoE patients with achalasia [128]. The injury caused by mast cells to nitrergic nerves and ICCs supported their role in esophageal motility disorders [129]. Our team compared the LES muscle specimens from 116 achalasia patients and controls [130], demonstrating a significant elevation in mast-cell infiltration within the LES muscle, accompanied by a decrease in ICCs and neuronal degeneration. These observations proved that mast cells might serve as crucial mediators in the pathogenesis of achalasia, which further deepens the understanding of the inflammatory response in the etiology of achalasia.

Macrophages

Macrophages, also known as histocytes, differentiate from monocytes after penetrating blood vessels [131]. As key players in the immune system, macrophages are increasingly recognized in gastrointestinal motility diseases. Recently discovered neuroimmune mechanisms—particularly the interaction between enteric neurons and gut macrophages—have emerged as a crucial factor for regulating intestinal peristalsis [132–134]. Gut macrophages were initially described in the early 1990s by Mikkelsen [135] and were defined as a macrophage subtype residing in the myenteric plexus associated with both ICC and enteric neurons [133, 136, 137]. They constitute a highly specialized subset of cells. Different gut-macrophage subsets occupy specific intestinal niches, each with a distinct transcriptome tailored to meet the functional requirements of their tissue microenvironment [136]. For instance, lamina propria macrophages maintain gut homeostasis by protecting the epithelial barrier and phagocytosing luminal antigens [137–139], while deeper muscularis external macrophages support enteric neurons. When inflammation in the muscularis activates resident macrophages, they will impair neuromuscular contractility and attract inflammatory cells [140, 141].

Macrophages exhibit a consistent and regular distribution within the serosa and at the site of Auerbach’s plexus [141]. The intricate interplay between muscularis macrophages and neurons plays a pivotal role in regulating intestinal motility and fostering tissue protection amidst injury and infection [136, 142]. It is reported that vagus-nerve stimulation interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages, improving postoperative ileus [143], indicating that such a complex interaction can also contribute to tissue damage in gastrointestinal motility disorders. Other related gastrointestinal motility diseases, such as inflammatory bowel disease (IBD) [144, 145] and irritable bowel syndrome (IBS) [146, 147], also support the idea that macrophages are associated with gastrointestinal motility disorders. Besides, macrophages and mast cells are recognized as crucial players in innate immune defense, contributing to the production of pro-inflammatory mediators during classical activation, which may potentially induce tissue damage [148].

In order to further understand the functional pathways of macrophages, our team has discovered through zoological experiments that the number of lamina propria macrophages increased after antibiotic exposure, with the TLR4-MYD88-NF-κB pathway upregulated, and the production of TNF-α, IL-1β, and IL-6 increased in the antibiotic-treated mice [149]. This coincides with the mediation pathway of inflammatory factors. However, previous studies have indicated no difference in the proportion of different macrophage subsets in achalasia. Our team employed single-cell RNA sequencing (scRNA-seq) to analyse LES tissues and blood samples from individuals with achalasia [150]. We found that C1QC+ macrophages exhibited transcriptional similarity to microglia within the central nervous system and manifested a phenotype indicative of neurodegenerative dysfunction in the context of achalasia, which meant that they potentially facilitated the disruption of immune-cell equilibrium and induced neuronal impairment.

Generally speaking, gut macrophages originate from monocytes, occupy specific ecological niches, and regulate intestinal motility through neuroimmune interactions with intestinal neurons. Dysregulation can lead to gastrointestinal motility disorders. Similar subgroups were also discovered in our team’s recent single-cell sequencing, suggesting a key role in the etiology of achalasia. Considering their close connection with the brain–gut axis, research on the specific ways of their phagocytic synapses and related molecular pathways has become a new hotspot, requiring large-sample single-cell sequencing and proteomics experiments.

Lymphocytes

T and B cells occupy central roles in adaptive immunity, distinguished by their unique surface receptors. T cells recognize antigens via major histocompatibility complex class I or II molecules, triggering immune responses or targeting infected cells. T cells are categorized into cytotoxic CD8⁺ T cells and CD4⁺ T cells. Antigen-presenting cells present antigens to T helper cells (Th cells), which can be subdivided into Th1, Th2, Th17, and Th22 subsets. Among these, Th1, Th17, and Th22 subsets exert pro-inflammatory effects, while Th2 cells mediate anti-inflammatory responses. B cells, on the other hand, are responsible for secreting antigen-specific antibodies.

In the inflammatory response, there are numerous types of T cells and it is particularly crucial to identify the exact pathogenic subgroups. Previous studies using immunohistochemical staining have revealed that, in achalasia tissues, CD3⁺, CD4⁺, CD25⁺, and CD8⁺ T lymphocytes were dominant, while CD20⁺ B lymphocytes and eosinophils were also present to a lesser extent [11, 30, 62, 116, 151]. A current debate revolves around whether CD4⁺ or CD8⁺ T cells predominate in achalasia. Our team utilized an scRNA-seq technique and found that tissue-resident memory T cells, especially ZNF683⁺ CD8⁺ tissue-resident memory T cells and XCL1⁺ CD4⁺ tissue-resident memory T cells, were significantly expanded in achalasia and localized around the myenteric plexus in the LES tissue of achalasia [150, 152, 153]. Inflammation increases with disease progression, which provides a reference basis for the classification of subtypes of achalasia. In a similar discussion on the inflammatory environment, Muller et al. focused on the differential diagnosis between achalasia and CD4⁺ T-cell-predominant lymphocytic esophagitis [154], which is in contrast to the finding of our team in another experiment, which showed that CD8⁺ T cells might be the key pathogenic subpopulation of achalasia [152, 153].

Regarding regulatory T cells (Tregs), regulatory B cells (Bregs), and regulatory plasmacytoid dendritic cells, Tregs play a crucial role in regulating the natural progression of protective immune responses, thereby limiting tissue damage and autoimmunity. Sodikoff et al. reported that Foxp3 [155], a defining marker of Tregs, was observed at a higher percentage in esophageal smooth muscle tissue from patients with type III achalasia, followed by types I and II. Additionally, a greater degree of ganglion-cell loss was detected in type I compared with type II specimens, supporting the hypothesis that type I achalasia represents a progression from type II achalasia, which was consistent with our team’s finding [150]. In addition to Tregs, Bregs also critically influence the inflammatory process of achalasia. They secrete anti-inflammatory cytokines such as IL-10 and IL-35, and transforming growth factor-β (TGF-β), as well as inhibitory molecules, serving as negative regulators of the immune system. Notably, biopsies taken from the myenteric plexus of patients with achalasia revealed a significantly higher percentage of IL-10-producing B cells compared with tissues obtained from a control group [11]. Similar results can also be observed in the pro-inflammatory and anti-inflammatory balance, suggesting that T cells and B cells may also achieve a balance in inflammation regulation.

Although there are numerous studies on T cells and B cells, only a few can provide feedback for clinical practice at present. Future research directions include investigating whether indicators such as the dominance of T-cell subsets and the levels of Treg/Breg can be used for subtyping achalasia and predicting disease progression. Furthermore, the specific interaction networks between immune cells and between the immune and neural systems also deserve more attention.

Cytokines, chemokines, and complements

Cytokines, chemokines, and complements play pivotal roles in inflammation. Cytokines regulate immune responses, chemokines guide white blood cells to infected areas, and the complements promote inflammatory processes and enhance immune-cell function. They work together to maintain immune balance and protect from infections. Abnormalities in their function can contribute to deregulated inflammatory responses and disease development. In a recent research review by our team [122], cytokines and inflammatory factors were divided into pro-inflammatory and anti-inflammatory pathways.

Pro-inflammatory factors

The expression of interleukin-22 (IL-22) has been observed to be increased in the tissue and circulating cells of individuals with achalasia [11]. IL-22 initiates innate immune responses against gut and respiratory pathogens while regulating antibody production. In achalasia samples, the percentage of IL-22⁺ cells was significantly higher than those in controls, with higher numbers in type II and III achalasia than in type I. As a regulator of antibody production, IL-22 is synthesized by the Th-cell subsets of Th22 and Th17. The Th22 cells differentiate from naive T cells in response to TNF-α and IL-6 signals, and subsequently synthesize and secrete IL-26 and IL-22 [156]. IL-6 levels were also significantly elevated in achalasia patients [152, 153]. Besides, IL-17, a key mediator of inflammatory autoimmune diseases, enhances immune-cell recruitment, activation, B-cell functions, and pro-inflammatory cytokines. The IL-17A⁺-cell frequency was higher in achalasia patients, particularly in types II and III. The percentages of interferon (IFN)-γ cells were also elevated in achalasia patients, with higher levels in types II and III [11].

Our team recently analysed serological data from 47 achalasia patients and 47 healthy controls, focusing on 51 cytokines and chemokines [157]. We observed significantly elevated levels of multiple cytokines in the achalasia patients [122], including IL-17, IL-18, IFN-γ, monokine induced by gamma, platelet-derived growth factor-BB, IFN-γ-induced protein-10, and stem cell growth factor (SCGF)-B [158, 159]. Notably, type III achalasia patients exhibited higher levels of IL-2Ra, IL-18, monokine induced by gamma, IFN-γ, SDF-1a, eotaxin, platelet-derived growth facto-BB, IFN-γ-induced protein-10, monocyte chemoattractant protein 1, and TNF-related apoptosis-inducing ligand compared with types I and II [160].

From a genetic perspective, the impact of these chemokines can also be supported. The interleukin-23 receptor (IL-23R) gene, situated on chromosome 1p31, encodes for the heterodimeric subunits of the IL-23R complex [161]. Multiple studies have demonstrated that IL-23R is implicated in both inflammatory and chronic autoimmune disorders [162], as well as IL-33 gene variants [163], suggesting that the IL-gene family may serve as a risk factor for the development of achalasia.

A pivotal report [164], marking the first genome-wide expression profiling of mRNA in achalasia patient tissue samples, has revealed the pivotal roles of toll-like receptor (TLR)-4 and IL-18. TLR-4, through its interaction with IL-18, is hypothesized to trigger a downstream signaling cascade that prompts the secretion of pro-inflammatory cytokines and chemokines. Additionally, genes integral to innate defense against pathogens, such as C–X–C motif chemokine ligand 17 (CXCL17), immunoglobulin heavy constant alpha 1 (IGHA1), and IGHA2, were downregulated in achalasia patients [165, 166]. CXCL17 encodes a homeostatic, mucosa-associated chemokine involved in innate immunity and tumor progression acceleration. IGHA1 and IGHA2 are protein-coding genes that may function in local infection defense and the prevention of foreign antigen access to the immune system.

Anti-inflammatory factors

TGF-β regulates cellular growth, proliferation, differentiation, inflammation, collagen synthesis, and apoptosis. In esophageal biopsies from achalasia patients, the expression of TGF-β1⁺ cells was elevated [11], especially in types I and III achalasia. IL-4 suppresses the production of TNF-α, IL-1β, IL-6, and IL-17A, and the proliferative and differentiative processes of Breg, and is primarily synthesized by Th2 cells. IL-4⁺ cells were higher in achalasia patients, particularly in types II and III. IL-13, similarly to IL-4, regulated type I collagen genes and was involved in fibrosis, with an increase observed in type II achalasia [11].

Complements

The complement system is a group of soluble proteins present in human serum and body fluids, as well as a group of membrane-bound proteins and complement receptors present on the surface of blood cells and other tissue cells. The complement system belongs to innate immunity and plays an important role in the body’s defense and in mediating immune pathological damage. Only a limited number of studies have hitherto delved into alterations in the complement system in achalasia. Im et al. performed the first proteomic analysis of serum samples from achalasia patients [167], revealing an upregulation of complement proteins. These upregulated proteins encompassed complement C4B5, C3 [152], cyclin-dependent kinase 5, transthyretin, and alpha-2 macroglobulin. Nevertheless, these investigations were constrained by the use of low-resolution techniques and the inclusion of small sample sizes, and none had simultaneously analysed both serum and muscle proteomics.

More recently, a 4D label-free proteomic investigation revealed a upregulation of complement proteins [168], including C1q, C1s, C2, C3, C4-A, C4-B, C5, C6, and C9, in achalasia patients. Studies have established that damaged myelin or autoantibodies can bind and activate C1. Once activated, C1 initiates the classical complement pathway, which can indirectly harm neurons by activating inflammatory cells and directly through the formation of the membrane attack complex [169]. Further research into the role of the complement and coagulation cascade pathways in achalasia is essential, as it could potentially yield a novel therapeutic target for patients with this condition.

Autoantibodies

According to recent reports, individuals with achalasia exhibit a significantly higher risk of developing autoimmune conditions that is ∼3.6 times greater than that of the general population [170], suggesting a correlation between achalasia and autoimmunity. Furthermore, elevated levels of circulating IgG antibodies targeting the myenteric plexus in most patients with achalasia suggest a potential role for autoantibodies in the pathogenesis of this disease.

A key question in the medical community is whether autoantibodies are the causative agent of achalasia or merely a symptom of the underlying condition. Notably, autoantibodies against M2-muscarinic acetylcholine receptors have been identified in patients with achalasia who also suffer from chronic Chagas disease [171]. In this context [85], anti-M2R antibodies were found to be significantly more prevalent among chagasic patients with achalasia (86%) compared with non-achalactic patients (28%), patients with idiopathic achalasia (21%), or healthy volunteers (0%). Recently, innate immune cells and extracellular proteolysis have been added to the classical paradigms of autoimmunity [172]. One enzyme of extracellular proteolysis, matrix metalloproteinase-9 (MMP-9), has been implicated in autoimmune diseases linked to achalasia [173]. The findings revealed a substantial elevation in the ratios of MMP-9/MMP-2 and activated MMP-9/pro-MMP-9 in the sera of patients with achalasia. Notably, many achalasia patients also possess autoantibodies targeting other organs [174], such as thyroid or gastric parietal cells, or anti-nuclear antibodies. Other autoimmune diseases, such as myasthenia gravis [175], inclusion body myositis [176], and Sjogren’s syndrome [177], were also reported. These reports suggested a potential link between achalasia and autoimmunity. However, the diagnosis of an autoimmune disease requires meeting three criteria: (i) direct evidence stemming from the transfer of the disease via antibodies, (ii) indirect evidence derived from the replication of the autoimmune condition in an animal or genetic model, and (iii) the detection of autoimmune T cells within the target organ. Therefore, more studies are needed in this field.

Inflammation and autoimmune responses have been deeply involved in the pathogenesis of achalasia. As the most extensively studied and most well-characterized pathogenic mechanism at present, inflammatory responses have shown significant experimental results both in clinical sample testing and animal experimental models. As the final factor inducing neuronal damage (Fig. 1), current research places more emphasis on dissecting the details of inflammatory responses at the molecular mechanism level, which is the core pathogenesis of achalasia. In addition, the inflammatory response involves a wide range of inflammatory factors in large quantities. Related studies can lay the foundation for future achalasia molecular targeted drug therapy. However, research on the diagnostic strategies and risk interventions for achalasia remains limited.

Most of the targeted drugs currently used in clinical practice directly reduce LES tension: endoscopic botulinum toxin injection inhibits cholinergic neurotransmitters, providing good short-term relief, but requires multiple administrations and is only applicable to elderly patients or those with comorbidities who are not suitable for POEM surgery [178]. Calcium antagonists provide short-term relaxation of the LES before meals, but their side effects and tolerance limit long-term application; PDE-5 inhibitors can significantly reduce the resting pressure of the LES in small samples and systematic reviews, and are still used in an exploratory and individualized manner [179].

The current forward-looking directions are as follows: one is causal intervention based on immunity-antiviral mechanisms, although there is still a lack of clinical evidence [180]; the other is the regeneration and replacement of the enteric nervous system. Multiple preclinical studies and reviews suggest that transplanting intestinal neural stem cells may restore neuronal and dynamic functions, but it is still at the stage of animal experiments or proof-of-concept studies [181]. Additionally, bioinformatics research has inferred from multiple cohort data that different subtypes of achalasia may have different drug targets, providing a candidate list for future “subtype-molecular stratified treatment” [182].

Psychological factors

Numerous systematic psychological studies have demonstrated that psychological and mental factors play a crucial role in gastrointestinal diseases such as IBD and IBS [183–185]. There have been reports indicating a higher occurrence of mental disorders within a year following the diagnosis of achalasia, such as depression, hypervigilance, and anxiety [186–188]. Our team confirmed this association and found that psychological distress was more prevalent in patients with achalasia compared with healthy controls [189]. Notably, the performance of POEM appeared to effectively alleviate psychological distress, indicating a positive impact on the patients’ psychological health. In addition, the severity of symptoms was significantly correlated with mental health status and higher anxiety scores were associated with more pronounced chest pain.

The exact role of psychological factors in the pathogenesis of achalasia remains unclear, with current studies presenting varying hypotheses. Some researchers postulated that stress triggered the aberrant secretion of corticotropin-releasing factor [190] or thyrotropin-releasing hormone [191], leading to inappropriate autonomic innervation of the gastrointestinal tract. Others thought that stress might alter the composition of the microbiota [192], elevating the susceptibility to inflammatory stimuli within the gastrointestinal tract and ultimately leading to perturbations in enteric neuronal function. In order to better elucidate the underlying mechanisms in this area, further psychological studies and fundamental experiments are urgently needed.

Gut microbiota

Gastrointestinal motility, a crucial physiological indicator, governs the digestion and assimilation of nutrients and its regulation is influenced by diverse factors including the enteric nervous system, the immune system, gut hormones, and gut microbiota [193–195]. The bacterial component within the gastrointestinal tract exhibits a relatively stable composition, consisting primarily of four major phyla: Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria.

It has been reported that bacteria can modulate gut motility through interactions with the enteric neurons and immune system [196], and intestinal stromal cells, such as ICCs and enteric glial cells, which may mediate the interactions between gut microbes and the enteric nervous system [197, 198]. In fact, disturbances in the gut microbiota, unrestrained immune reactions to harmful agents, and compensatory modifications within the enteric nervous system are pivotal contributors to the etiology of a variety of gastrointestinal conditions. Many functional gastrointestinal disorders, such as IBS [199–201], IBD [202–204], and postoperative ileus [205], are associated with the gut microbiota.

Given the strong connection between the pathogenesis of achalasia and enteric neurons, it is reasonable to hypothesize that the esophageal microbiota may influence the LES. Recently, our team used 16S rRNA sequencing and found that, in individuals suffering from achalasia [149], the diversity of the esophageal microbiota was diminished, featuring an abnormal predominance of Gram-negative bacteria, constituting a microbial profile known as type II microbiota. Additionally, a notable decrease in the relative abundance of the Rhodobacter genus has been observed among achalasia patients. Another study performed the microbial composition analysis of the esophagus in achalasia patients, revealing that Fusobacteria collectively accounted for > 95% of the total phyla across all groups [206]. At a more specific genus level, Streptococcus emerged as the most prevalent genus in all patient groups. Among the Firmicutes, the discriminating enriched genera in achalasia patients were Lactobacillus [207] and the abundance of Lactobacillus was found to correlate with the severity of the condition, suggesting a potential role in the disease progression [208].

Additionally, the bacterial flora in achalasia patients’ stasis fluid comprises microorganisms from the normal mouth and oropharynx microbiota, thriving in a nutrient-rich, neutral-pH environment with low oxidation-reduction potential, conducive to anaerobic growth [209]. The mechanism of the esophageal microbiota in achalasia was reported to be closely related to the Th17 immune response [210]. There is no doubt that the intestinal microbiota is currently a research hotspot for various gastrointestinal dynamic diseases and the signaling-pathway mechanism of microbiota-immune-neural remains to be further discovered.

Gut–brain axis

The gut–brain axis refers to the intricate bidirectional communication network between the gastrointestinal tract and the central nervous system (CNS). This interplay involves the nervous, immune, and endocrine systems, facilitated by neural pathways, hormones, and immune messengers. The gut–brain axis modulates various physiological and behavioral functions, including digestion, mood, stress response, and cognitive processes. It has been documented that gastrointestinal-tract motility diseases frequently coincide with a growing prevalence of CNS disorders [211]. The influence of the brain–gut axis on gastrointestinal motility diseases has been reflected in IBD [144, 212], IBS [201, 213], and functional dyspepsia [214].

Recently, researchers have found the relationship between achalasia and CNS diseases [215]. Using resting-state functional magnetic resonance imaging, they investigated the brain features in patients with achalasia compared with healthy controls. The results showed that patients with achalasia exhibited enhanced functional connectivity between the nucleus ambiguus and several cortical regions, including the right precentral gyrus, the left postcentral gyrus, and the left insula. This indicated that abnormal resting-state functional connectivity and regional brain activity were found in achalasia patients, suggesting that the etiology of achalasia may have a certain connection with the brain–gut axis.

Regarding the mechanisms by which the brain–gut axis may influence achalasia, several reasonable hypotheses can be posited. First, the vagus nerve—a primary pathway of the brain–gut axis—might be implicated in achalasia due to its role in regulating gastrointestinal motility [216, 217]. Besides, immune mechanisms from microbiota to gut–brain signaling also play a role. The brain–gut axis can modulate inflammatory responses, with inflammatory mediators potentially contributing to the development of achalasia [218, 219]. Furthermore, the gut microbiota generates metabolites that influence the physiology and behavior of the CNS and enteric nervous system. Studies have indicated that certain bacteria produce neurotransmitters, such as noradrenaline, dopamine, and gamma-aminobutyric acid [220, 221]. Finally, stress and other psychological factors, which can be communicated with ENS through the brain–gut axis, might also contribute to the onset or exacerbation of achalasia [189].

The advent of brain imaging has enabled the visualization of these reciprocal interactions, revealing that gut stimuli can activate crucial brain areas for emotion regulation. There are other neurological diseases occurring simultaneously with achalasia, such as Parkinson’s disease [222], Allgrove syndrome [66], Hirschsprung’s disease [223], Wernicke’s encephalopathy [224], multiple sclerosis [225], and brain-stem infarction [226], indicating the potential relationship between the gut–brain axis and achalasia.

Although many causes of achalasia have been summarized in this review, the relative weights between them vary. Among them, the evidence for genetic factors, viral infections, and inflammatory responses is the strongest. This has been supported by clinical studies and animal experiments, and the internal pathways involved have also been thoroughly investigated.

The evidence for the esophageal microbiota, brain–gut axis, and psychological factors proposed at the end is weaker than that for the first three causes. It is based on other gastrointestinal motility disorders and certain clinical statistics as speculations. The evidence is relatively preliminary and there is also a lack of large-sample verification, cell models, and animal models. More strong evidence is needed to verify this phenomenon and explore the mechanism.

However, although the evidence for the three etiologies of esophageal flora, brain–gut axis, and psychological stress is not strong, it puts forward new possible mechanisms and provides new ideas for future research. Based on the existing clinical phenomenon, these factors can only be proved to be associated with achalasia and the existing clinical evidence cannot support them as the decisive factors of etiology.

Conclusions

Achalasia—a primary esophageal motility disorder—remains elusive in its epidemiology, diagnosis, and treatment. The pathogenesis of achalasia remains unclear, necessitating further investigation. Studies have suggested that neurodegeneration in the LES due to the loss of inhibitory neurons can be the major factor. Genetically predisposed individuals may be more susceptible to these pathogenic factors, such as psychology, gut microbiota, and the gut–brain axis. These factors will trigger autoimmune and inflammatory responses against inhibitory neurons in the LES. Consequently, the loss of inhibitory neurons in the esophageal myenteric plexus causes the failure of the LES relaxation and esophageal peristalsis.

We innovatively reviewed the psychological factors, the gut microbiota, and the brain–gut axis as new perspectives in the pathogenesis of achalasia and conducted a clearer critical assessment of the relative importance of each possible pathogenic mechanism. However, the absence of reliable cell and animal models has hindered the validation of this hypothesis and the elucidation of precise biological pathways. Furthermore, the clinical heterogeneity observed in achalasia underscores the need for studies that delve into the varying pathogenic mechanisms between different subtypes.

Funding

This study was supported by the National Natural Science Foundation of China [grant numbers 82170555, 82270569, and 82200600], Shanghai Academic/Technology Research Leader [grant number 22XD1422400], Shuguang Program of Shanghai Education Development Foundation and Shanghai Municipal Education Commission [grant number 22SG06], and the Shanghai “Rising Stars of Medical Talent” Youth Development Program [grant number 20224Z0005].

Conflicts of interest

None declared.