The functional landscape of the appendix microbiome under conditions of health and disease

The human appendix, once thought to be a vestigial organ, has newly attracted interest for its possible function in maintaining healthy gut microbiota [1, 2]. The term "appendix" comes from the Latin word "appendere," which means "to hang upon" [3]. Historically, the appendix was named because it hangs on to the cecum, which resembles a little appendage suspended from the large structure. This etymology reflects the appendix's presumed function as a secondary addition to the gastrointestinal tract [4, 5]. Research suggests that the appendix functions as “sanctuary” for beneficial gut bacteria, protecting them and aiding in their replenishment after disruptions, such as diarrheal illness [6]. This bacterial reservoir may be crucial for restoring gut flora, which is essential for various bodily functions, including immune response and digestion [6, 7].

The appendix has evolved in an independent way across different species, including rodents, primates,, and marsupials [8‐11]. The recurrence of evolution suggests that the appendix has important functions, rather than being a vestigial organ [9]. Research recommends that the appendix has followed an independent evolution in various mammals, which is a clue for its functional role [9, 12]. It appears that the appendix has been present as an element of mammals’ digestive system for a very long time of at least 80 million years [13, 14]. This is a clue for a selective advantage by this organ [12].

In a healthy state, the AM helps keeping a balanced gut flora, which is essential for the overall body function [15]. In the case of a disease, alterations in AM composition can contribute to or exacerbate conditions like acute appendicitis, which is linked to lowered microbial diversity and a surge in pathogenic bacteria [6, 16]. Furthermore, appendix removal (appendectomy) has been linked to changes in the GM and is believed to cause long-term impacts on human health, including an elevated risk for cancer and certain neurological disorders [7, 17‐20].

The gut-brain axis (GBA) connects the enteric and central nervous systems, involving hormonal, immunological, and neuronal pathways that together regulate gastrointestinal (GI) function, mood, and cognitive processes [21, 22]. It can be considered a bidirectional network of communication. A crucial component of this axis is the microbiome, consisting of trillions of microorganisms in the gastrointestinal (GI) tract that have a profound impact on the host's health and disease states [23]. Emerging studies suggest that the AM may influence the GBA. The GM can directly impact brain function and behavior, potentially affecting neurodevelopmental and psychiatric disorders [24, 25]. Specific microbial species in the AM are involved in neurotransmitter production. For instance, Lactobacillus and Bifidobacterium species produce GABA (gamma-aminobutyric acid), which helps regulate anxiety and stress responses [26]. E. coli produces serotonin, an important molecule [27]. Enterococcus faecalis produces dopamine, essential for reward and motivation pathways in the brain [27]. Clostridium species set free SCFA (short-chain fatty acids) like butyrate. These exhibitanti-inflammatory effects and can influence brain health [28]. As a microbial reservoir, the appendix may serve a vital function in regulating the GBA, particularly during periods of gut microbial imbalance [29]. The production of neurotransmitters by specific microbial species highlights the complex relationship between the gut and brain, with imbalances potentially resulting in neurological disorders. For instance, reduced GABA and serotonin levels are associated with depression and anxiety disorders [26], while alterations in dopamine production are linked to Parkinson's disease [27]. Furthermore, changes in SCFA production due to AM dysbiosis can impact cognitive function and contribute to Alzheimer's disease [28]. Santos et al. have found that stress is a potentiator of symptom severity in these disorders via the brain-gut-microbiota axis. This highlights the significant clinical implications of understanding the interaction between chronic psychological stress, gut microbiota, and the GBA in mental health and digestive diseases [30]. These insights underscore the significance of the AM in maintaining gut-brain communication and highlight its potential therapeutic implications for neurological health.

The precise mechanisms through which the AM interacts with various bodily functions are still being explored. However, it is hypothesized that the appendix may contribute to health by modulating the immune system, as it contains lymphoid tissue that supports the growth of beneficial gut bacteria. It is also likely that the appendix influences health by acting as a reservoir for bacteria that produce neurotransmitters or their precursors, potentially influencing mood and cognitive functions [22, 23, 31].

The AM is gaining recognition to impact health and diseases. Its potential influence on health offers new insights into conditions ranging from GI disorders and cancers to mental health issues. As research continues to resolve the complexities of the microbiome and its relations with the human body, the appendix may no longer be seen as a redundant organ but as a vital contributor to overall well-being.

In this review, the authors discuss factors affecting AM function and composition, diseases associated with AM dysbiosis (a microbial imbalance in the appendix or gut microbiome), and its therapeutic prospects. Throughout the review, the term “GM” was used frequently, as the GM and AM share similarities at the phylum and genus levels, although their composition differs. Since few studies have addressed the health impacts of the AM, this review offers a comprehensive overview, covering nearly all aspects of the relationship between the GM and AM, and it thereby provides valuable insights into their roles in health and diseases.

This review thoroughly examines the determinants of AM function and composition, AM dysbiosis disease, and its therapy. An extensive literature search and meta-analysis was conducted with well-established databases and tools such as Web of Science, PubMed, Scopus, and Dimensions AI tools (Fig. 1) [32, 33]. The search was filtered with 2020 to 2024 published studies that examined the appendix, gut microbiome (GM), and AM functions in health. Some specific keywords like “Appendix microbiota”, “Gut microbiota”, “Appendix microbiota and colorectal cancer”, “Appendicitis and inflammatory bowel disease”, and “Appendicitis and neurological disorder” were used to search relevant articles.

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To ensure the quality and relevance of included studies, specific exclusion and inclusion criteria were also used. The articles were screened based on their research strengths, peer-review status, and relevance to our review focus. Specifically, studies that discussed the function of the appendix in gut immunity and health, explored the impact of the appendix on disease progression, or investigated therapeutic applications of the AM were given importance. Articles that published between the years 2020–2024 received priority to capture the recent trends in the field. Non-peer reviewed studies, non-AM or GM specific articles, published before 2020, and duplicates/preprints were excluded from analysis.

Data extraction involved meticulous review of the abstracts and texts of the included articles to ensure their relevance and quality. Published data were gathered on different themes, including the appendix as a reservoir for defensive bacteria, impact of appendix removal (appendectomy) on diversity of gut microbiota, immunological functions of the appendix, and therapeutic application of the AM in various diseases. The influence of the appendix on disease progression was also a focal point of the analysis.

To judge the effect of AM on public health, a bibliographic analysis of recent research was conducted using VOSviewer (Fig. 2). The analysis was conducted with VOSviewer version 1.6.20 and Dimensions research publication database with 152 published articles in the period from 2020 to 2024 on AM and related diseases [32, 34]. The analysis was done with 100 keywords extracted from the publications and yielded four clusters in key words.. The network shows the geographical and time spread of the research, with teamwork in addressing AM and its impact..The findings of the network analysis and literature review were combined to give an overview of the appendix's role in gut immunity and health. The analysis brought out the transformation of the appendix from a mysterious organ to an essential component of gut health. It addressed the appendix as a healthy reservoir that is strong, the impact of disruption of the appendix on disease occurrence, and the therapeutic value of the AM in optimizing health outcomes.

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The composition of AM and its function is impacted by various factors like diet, disease, antibiotic use, and chemotherapy (Fig. 3) [35‐37]. By knowing these influences, targeted interventions can be developed, such as dietary modifications or probiotic supplementation, to maintain or restore a healthy AM [24, 38]. As research in this field advances, it will provide further insights into the complex interactions between our lifestyle, health, and the microbial communities within us.

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The AM is highly important both for gut health and immune function. Changes in the AM can lead to diseases such as acute appendicitis, IBD, back pain, obesity, diabetes, cancer, and so on [52, 79, 80]. Figure 4 shows the microbial compositional changes that cause different disease development, whereas Fig. 5 shows the effects of gut dysbiosis in different physiological outcomes.

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The GBA is an intricate bidirectional communication network that links the GI tract with the central nervous system (CNS). This dynamic interplay involves complex interactions through neural, immune, and endocrine pathways [143, 144].

The GM contributes to CNS homeostasis by modulating the immune system and regulating the production of various molecules and metabolites [38, 145]. These metabolites, such as SCFAs produced by Bacteroidetes and Firmicutes, neurotransmitters, and other signaling molecules, can cross the blood–brain barrier and impact brain function directly [146, 147]. Moreover, the immune system's interaction with the GM helps maintain a balanced inflammatory response, which is crucial for preventing neuroinflammation [148, 149].

Neurological disorders (NDs) such as depression, schizophrenia, Parkinson disease (PD), Amyotrophic lateral sclerosis (ALS), autism spectrum disorder (ASD), epilepsy, and migraine have been linked to dysbiosis in GM [150]. While working with the C57BL/6 J mouse model, depletion of Lactobacillus and enrichment of Akkermansia were associated with neuroinflammation [151]. According to a recent study, stress may have caused gut dysbiosis, which in turn may have increased the abundance of conditional pathogens like Enterococcus, Vagococcus, and Aerococcus, as well as pathogens like E. coli and Shigella [152]. Alcoholism patients' GM differed significantly from healthy adult patients' GM. Firmicutes greatly increased in number whereas Bacteroidetes significantly declined. Mice receiving fecal microbiota from alcoholism patients displayed behaviors resembling anxiety and depression, a reduction in socialization, a spontaneous favor for alcohol, and a reduction in brain-derived neurotrophic factor (BDNF) when compared to mice transplanted with fecal microbiota from healthy male adults [46]. It's interesting to note that individuals with schizophrenia typically experience GI complications, have a greater frequency of intestinal barrier failure, and exhibit higher bacterial translocation [153]. The genera like Succinivibrio, Megasphaera, Collinsella, Clostridium, Klebsiella, and Methanobrevibacter were significantly higher in schizophrenia, whereas Blautia, Coprococcus, and Roseburia were abundance in healthy controls. Patients with first experience of schizophrenia significantly lowered numbers of Bifidobacterium, E. coli, and Lactobacillus [154]. A similar result was reported by other studies that showed a correlation between Succinivibrio and Corynebacterium and the degree of schizophrenia symptoms [155]. Another study reported an increase in Anaerococcus and a decrease in Haemophilus, Sutterella, and Clostridium in schizophrenia [156].

PD showsdegeneration of dopaminergic neurons. GM alterations may precede the onset of motor symptoms in PD. Interventions aimed at restoring microbial balance could potentially slow down disease progression [157, 158]. Increased genus Akkermansia, which promotes intestinal permeability, and decreased SCFA-producing bacteria of the genera Roseburia and Faecalibacterium are frequently linked to PD [159]. The most consistent change in the GM composition in PD was found in a more recent meta-analysis of eleven case–control studies, which revealed decreased genus Faecalibacterium and Lachnospiraceae family and increased genera Lactobacillus, Akkermansia, and Bifidobacterium [160]. Shen et al. reported similar changes in a meta-analysis involving fourteen studies. Reductions in Prevotellaceae, Faecalibacterium, and Lachnospiraceae were observed in the analysis, whereas increases in Bifidobacteriaceae, Ruminococcaceae, Verrucomicrobiaceae, and Christensenellaceae were noted [161]. Rodent models of PD have shown a similar pattern of gut dysbiosis [162]. Conflicting findings regarding the role of the appendix and PD been reported by recent studies. Park et al. found no link between appendectomy and risk change of PD in the Korean population, indicating that appendectomy did not determine the risk of PD [163]. In contrast, Wang et al. found out that appendectomy is associated with a lowered risk of PD, highlighting a potential protective effect of the surgical removal of the appendix [164]. Similarly, Jain et al. found no increased risk of PD following appendectomy, suggesting that the appendix is not a critical site for the development of PD pathology [165].Nakahara et al. described a significant phylogenetic difference in the gut microbiota between PD patients and healthy controls who had undergone appendectomy [166]. Their findings suggested a potential role of the gut microbiota, in particular the family Enterobacteriaceae, in the development of PD. The variable findings of these studies point to a variety of limitations and areas for future research. The studies were conducted in different populations, and this may be the reason behind the variable results. The studies were conducted through different methods, such as Cox regression analyses and Mendelian randomization [163, 164, 166], which may influence the results. Genetic susceptibility to PD may vary between different populations, and this might influence the relationship between appendectomy and risk for PD. Potential confounders such as age, sex, and underlying illness may not have been accounted for in all of the studies. Larger populations and more representative groups are needed to validate the results and a clearer causal link. Some studies, like Nakahara et al. are preliminary and must be validated by larger, more precise studies [166]. To bridge these gaps, subsequent studies should involve large-scale, multi-center studies with heterogeneous populations to account for both genetic as well as environmental heterogeneity. Standardized methods between studies would allow better comparison of outcomes. In addition, control of potential confounders is important to differentiate the true effect of appendectomy on the risk of PD. Long-term follow-up studies are required to ascertain the timing of the relationship between appendectomy and pathogenesis of PD. Mechanistic investigations should also study the precise mechanisms by which appendix and gut microbiota affect PD pathology.

ALS involves the degeneration of motor neurons. While less is known about the GM's role in ALS compared to AD and PD [167]. A study with ALS SOD1G93A mice yielded a reduction in Butyrivibrio fibrisolvens [162], which liberate butyrate. However, while working with humans no substantial changes were found in the ratio of Firmicutes:Bacteroidetes [168].

ASD is a collection of neurodevelopmental disorders. Concomitant pathologies in patients with ASD typically include gut dysbiosis, anxiety, depression, seizures, and other GI issues, like diarrhea, abdominal discomfort, and vomiting [169, 170]. It has been observed that people with ASD have higher biomass and less variation in bacteria [171, 172]. The most significant might be lower concentrations of Bacteroidetes, Actinobacteria, Proteobacteria, Prevotella, Corprococcus, and Veilonellaceae [171], combined with higher concentrations of Desulfovibrio spp., Sutterella spp., and Ruminococcus torques, or Clostridium spp. [173, 174]. Additionally, other authors reported an excess of Megamonas, and Candida spp., which were found to set freeammonia and other toxins that contribute to autistic behavior. They also saw a decline in the ratio Bacteroidetes:Firmicutes [174‐176]. Additionally, the onset of Asperger's syndrome was connected with a decrease of pro-inflammatory in Bifidobacterium spp. [177]. According to earlier research, patients with autism may benefit from butyrate-producing bacteria like Eubacterium, Ruminococcaceae, Erysipelotrichaceae, and Lachnospiraceae [178].

Epilepsy is a seizure disorder and aneurological disease. GM composition differs between drug-resistance and drug-responsive epilepsy patients. Compared to patients who experienced more than four seizures annually, those who experienced four or fewer per year displayed higher levels of Bifidobacteria and Lactobacillus [179]. Recent studies suggest a ketogenic diet to restore GM and protest epilepsy. The level of Parabacteroides and Akkermansia muciniphila was elevated by the ketogenic diet. Seizures were prevented when gnotobiotic cocolonization was enhanced with Akkermansia and Parabacteroides [180]. Moreover, probiotic supplementation of Lectobacillus and Befidobacterium reduced 50% of drug-resistance epilepsy [181].

A “leaky gut”-related increase in proinflammatory cytokines may contribute to the onset of migraine discomfort [182]. A significant correlation between GM and migraine was established. There was a notable enrichment of Firmicutes, particularly Clostridium spp., in the migraine group, whereas, the healthy controls had higher concentrations of advantageous bacteria, including Methanobrevibacter smithii, Bifidobacterium adolescentis, and Faecalibacterium prausnitzii [183].

The microbiome in the gut plays a very crucial role in protecting the host organism against numerous infections, including those caused by parasitic protozoa and fungi. The intestinal microbes regulate fat storage, stimulate epithelial cells, and influence brain and immune system development. All these interactions are significant in maintaining homeostasis and preventing infections [184, 185]. Intestinal protozoa-microbiome interactions emphasize the immunomodulatory effect of the intestinal microbiota on protozoan infection like Entamoeba histolytica, Blastocystis spp., Giardia duodenalis, Toxoplasma gondii, and Cryptosporidium parvum [184, 185]. The organization of intestinal bacterial communities can modulate the path of protozoan infection and the destiny of parasitic disease [186]. In parasitic illnesses associated with intestinal parasites, e.g. amoebic colitis or amoebic liver abscess due to E. histolytica or intestinal giardiasis due to G. duodenalis, interactions between intestinal bacteria and such parasites could affect parasitic or bacterial virulence. For instance, reduced abundance of Bacteroides, Clostridium, and Lactobacillus and increased abundance of Bifidobacterium is associated with amoebic colitis, while increased abundance of Prevotella copri is associated with amoebic diarrhea [187, 188]. Non-pathogenic parasitic protozoa also have the potential to alter intestinal microbiota diversity and provoke host immune responses. For example, asymptomatic colonization with Blastocystis spp. is associated with raised total diversity of intestinal bacteria and widespread changes of the dominant species within the gut microbiota. But colonization by Blastocystis hominis has been associated with reduced numbers of some markers of intestinal immunity, suggesting an anti-inflammatory environment within the intestine [184, 189, 190].

Current investigations explore the potential of fecal microbiota transplantation (FMT) to control immune response. There is evidence that FMT from protozoa-exposed donors can inhibit the immune response in a germ-free mouse model, demonstrating its potential to modulate immune responses and influence intestinal physiology [184, 188]. Specifically, gut protozoa such as Blastocystis hominis and Entamoeba histolytica can modulate the immunoinflammatory status and induce functional changes in the intestine by interacting with the gut microbiota [188, 191]. This downregulation of intestinal immune response includes decreased production of such proinflammatory cytokines as IL-6, TNF, IFN-γ, MCP-1, IL-10, and IL-12 in FMT-receiving mice from protozoa-exposed donor mice [188, 192]. Investigation into intelligent biological networks indicates a potential role through increasing anti-microbial resistance resilience by nutritional interventions. Enteric protozoan pathogenic infection effect on global health and the gut microbiome contribution towards infection is high-profile. Nutritional supplementation, including proteins, prebiotics, probiotics, synbiotics, and food formulations, is discussed as being a key part of recovery from dysbiosis and resistance to opportunistic pathogens [193]. Systems biology strategies are valuable tools to define the biochemical pathways of infection and recovery, allowing for better understanding of the impact of nutritional treatments on the gut microbiome and its role in protozoal infections [193, 194].

The imbalance of the gut microbiota, known as dysbiosis, can significantly impact the health status of the host. For example, Cryptosporidium spp. is an epithelial-lining-bound parasite that interacts with bacterial biofilms in the lining of the gut to cause behavioral adaptation in itself and neighboring bacterial populations [193, 195]. Studies have shown that Cryptosporidium growth is directly proportional to Pseudomonas aeruginosa biofilm maturation, indicating the complex interaction between parasites and bacterial communities [193, 195]. Similarly, Giardia infection may provoke alterations in gut microbiota, converting commensal bacteria into pathogenic forms and leading to increased virulence and inflammation [196]. In addition, protozoan infections can produce cross-organ effects. For instance, Cryptosporidium and Entamoeba have been known to cause opportunistic bronchial and pulmonary invasion, leading to respiratory conditions such as pneumonia, cough, and chest pain [193, 197]. Infections of the enteric type can lead to the host immune system releasing cytokines, which in turn cause inflammation and asthma. Exaggerated production of short-chain fatty acids (SCFAs) during infection potentially controls the immune system and dampens allergic responses, permitting regulatory T-cell differentiation [198]. These experiments highlight the interplay between the appendix, gut microbiota, and parasitological diseases. Microbiota manipulation has the potential to offer novel therapeutic approaches to the management of intestinal protozoan infections and related immune responses.

The AM may help to prevent and treat various diseases and could be leveraged for therapeutic approaches. They could be used to treat cancer, GI diseases, metabolic disorders, neurological disorders, cardiovascular diseases (CVDs), and so on [60, 115, 199‐201]. Methods that were applied to treat these diseases using appendix/GM are the use of prebiotics, probiotics, FMT (fecal microbial transplantation), engineered bacteria, phage therapy, RNA-guided gene therapy, and diet and metabolic modification (Fig. 7) [60, 199, 202‐205]. The main purpose of these methods is to reform the GM which ultimately impacts the overall health outcome.

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The AM emerges as a crucial contributor to human health, challenging conventional perceptions. It serves as a sanctuary for beneficial bacteria, crucial for gut health and immune system modulation. While direct evidence of the AM’s role in different diseases is still developing, the concept of using it as a therapeutic reservoir is promising and warrants further investigation. Research revealed that autoimmunity might be induced by FMT when transferred from autoimmune patients to healthy mice [89]. It is assumed that healthy subjects contain a balanced GM. When a transferred microbe by FTM causes autoimmune disease in healthy subjects, it means the transferred microbes might become dominant over the commensal flora. Issues like this could open a new era of research. Moreover, there are conflicting outcomes in research when working on the role of GM in some disease progression like PD. The conflicting evidence on the relationship between appendectomy and PD underscores the complexity of the disease and the need for a nuanced understanding of the GBA. Therefore, clinicians should consider appendectomy history when assessing PD, but more studies are required to clarify this relationship and explore therapeutic possibilities. Postbiotics could signify an advanced therapeutic approach if the microbial metabolites that are part of them can work synergistically on the target site (like the gut-vascular axis). In this instance, the intravenously supplied metabolites of bacteria might enter the bloodstream and provide direct action on, which is a novel indirect upgrade on vascular function brought about by the restoration of the intestinal microbiota’s homeostasis [211]. However, it is a promising advancement of science and could be applied to investigate other diseases as well.

Future research should focus on several key areas. There is a need for well-designed clinical trials and longitudinal studies to explore the role of AM in health and disease comprehensively. These studies should aim to understand the long-term effects of manipulating AM and its impact on various health conditions, including autoimmune diseases, neurodegenerative disorders, and metabolic syndromes. Research on gut fungi and parasites is significantly lacking compared to the gut bacteria. Future research should aim to elucidate the roles of gut fungi, parasites, and viruses, and their role in disease progression and their potential as therapeutic targets. Understanding the interactions between these microorganisms and the host immune system could lead to novel therapies for various diseases. The targeted change of microbiota by either restoringfunctions that are missing or eliminating functions which are harmful, may result in new methods to avoid or cure various ailments [21]. Future research promises innovative therapies harnessing the appendix's microbiota to enhance immune function and combat disease. Despite current limitations, particularly in human studies, understanding the appendix's complex symbiosis with the GM holds transformative potential for personalized medicine. As we unravel its complexities, the appendix represents the profound interplay between humans and their microbial counterparts, offering avenues for revolutionary healthcare advancements.