Authors: Prasant K. Jena (1Department of Pediatrics, Division of Pediatric Infectious Diseases, Guerin Children’s, Cedars-Sinai Medical Center, Los Angeles, CA, USA.; 2Infectious and Immunologic Diseases Research Center (IIDRC), Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA.), Moshe Arditi (1Department of Pediatrics, Division of Pediatric Infectious Diseases, Guerin Children’s, Cedars-Sinai Medical Center, Los Angeles, CA, USA.; 2Infectious and Immunologic Diseases Research Center (IIDRC), Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA.; 3Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.), Magali Noval Rivas (1Department of Pediatrics, Division of Pediatric Infectious Diseases, Guerin Children’s, Cedars-Sinai Medical Center, Los Angeles, CA, USA.; 2Infectious and Immunologic Diseases Research Center (IIDRC), Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA.)
Categories: Article, microbiota, Kawasaki disease, coronary artery, vasculitis, short-chain fatty acids, metagenomics, 16sRNA sequencing, antibiotics, metabolites
Source: Arteriosclerosis, thrombosis, and vascular biology
Authors: Prasant K. Jena, Moshe Arditi, Magali Noval Rivas
The intestinal microbiota influences many host biological processes, including metabolism, intestinal barrier functions, and immune responses in the gut and distant organs. Alterations in its composition have been associated with the development of inflammatory disorders and cardiovascular diseases, including Kawasaki disease (KD). KD is an acute pediatric vasculitis of unknown etiology and the leading cause of acquired heart disease in children in the USA. The presence of gastrointestinal symptoms in the acute phase of KD has been associated with an increased risk of treatment resistance and the development of coronary artery aneurysms. Studies report alterations in fecal bacterial communities of KD patients, characterized by the blooming of pathogenic bacteria and decreased relative abundance of short-chain fatty acid-producing bacteria. However, causality and functionality cannot be established from these observational patient cohorts of KD patients. This highlights the need for more advanced and rigorous studies to establish causality and functionality in both experimental models of KD vasculitis and patient cohorts. Here, we review the evidence linking an altered gut microbiota composition to the development of KD, assess the potential mechanisms involved in this process, and discuss the potential therapeutic value of these observations.
The gut microbiota comprises trillions of diverse microorganisms, such as bacteria, viruses, fungi, and archaea, which interact with the host to support various physiological processes, including regulating intestinal mucosal barriers, host metabolism, and immune responses^1,2^. These functions are mediated through the production of molecules, such as short-chain fatty acids (SCFAs), bile acids, trimethylamine N-oxide, tryptophan, indole, and gut peptides (glucagon-like peptide-1, PYY,..) among others^3^. Alterations in the intestinal microbiota composition, also called dysbiosis, may dysregulate their production, contributing to inflammatory and metabolic diseases, including cardiovascular diseases (CVD)^4^. Indeed, intestinal dysbiosis has been associated with the development of various CVDs, including atherosclerosis, hypertension, heart failure, abdominal aortic aneurysms, and coronary artery (CA) disease^5–8^. This association is not surprising, as several environmental and behavioral CVD risk factors, such as a poor diet, lack of physical activity, alcohol consumption, and smoking, can also impact the microbiota composition.
Kawasaki disease (KD) was first described in 1967 by Dr. Tomisaku Kawasaki, who documented 50 cases of a unique illness affecting children at the Tokyo Red Cross Medical Center in Japan^9^. Despite the disease being discovered over 50 years ago, there is still much to uncover and understand, from the unidentified etiological triggering agent(s) to the underlying immune mechanisms resulting in this systemic vasculitis and the potential long-term sequelae. KD is an acute febrile systemic vasculitis that predominantly affects children under five years of age^10^. KD has been reported globally, but the highest incidence occurs in Southeast Asian countries and among children of Asian ancestry. Indeed, Japan has the highest incidence rate, estimated to be 359 cases per 100,000 children under five years^11,12^. Boys are more often diagnosed with KD than girls, with a male-to-female ratio of 1.5:1^10–12^. The seasonality of KD, as well as the presence of KD geographical clusters, echo the spreading of infectious diseases, and these observations support the hypothesis that KD may result from one or multiple infectious pathogens acting on genetically susceptible children^13,14^. There is no specific diagnostic test; rather, KD diagnosis is primarily based on the presence of a persistent fever (more than five days) along with a combination of four of the following clinical skin rash, conjunctival injections, swelling, redness of the hands and feet, swollen lymph nodes (lymphadenopathy), and changes in the oral mucosa characterized by cracked lips and strawberry tongue^10^ (Figure 1A). Although the exact triggering agent(s) remains unidentified, the clinical and pathological features of KD, such as fever and acute onset of symptoms, indicate that both infections and altered immune responses are potential contributors, and an infectious pathogen may trigger the disease in genetically predisposed children^13–16^ . Without treatment, up to 25% of patients with KD may develop coronary artery (CA) aneurysms, potentially resulting in severe complications, such as myocardial infarction, ischemic heart disease, or sudden death^10^. The cardiovascular consequences of KD may persist into adulthood, with young adults who had KD in childhood at risk of long-term issues such as cardiomyopathy and myocardial ischemia, as well as a higher likelihood of premature atherosclerosis^17,18^. Intravenous immunoglobulin (IVIG) therapy reduces the risk of adverse outcomes to approximately 4% of KD patients^10,19^. However, the precise immune mechanisms behind IVIG’s positive effects remain unclear^20^. Additionally, approximately 20% of patients do not respond to IVIG, placing them at higher risk for developing CA aneurysms^19^.
Gastrointestinal (GI) symptoms such as abdominal pain, diarrhea, and vomiting are frequently observed at the onset of KD^21^. GI issues often make diagnosis and treatment more challenging and are associated with a greater likelihood of IVIG resistance and the formation of CA aneurysms^10,21,22^. Additionally, immunohistochemical analysis of intestinal biopsies from KD patients has shown an increased presence of HLA-DR^+^ cells and CD4^+^ T cells in the lamina propria, suggesting activation of the intestinal immune system^23^. Due to acute febrile symptoms similar to an infectious disease, children with KD are often administered antibiotics before a KD diagnosis is made^24–28^. Antibiotics profoundly disrupt the microbiota composition, and these perturbations can persist for long periods. Furthermore, antibiotics target and kill harmful bacteria and also eliminate bacteria that benefit the host’s physiology^29^, and antibiotic usage during childhood is associated with a higher risk of developing several immune-mediated diseases^30^.
Several studies have attempted to identify specific alterations in the composition of intestinal bacterial communities associated with KD^31–43^. However, while these studies offer hints and clues linking changes in the microbiota composition to KD development, there is not yet an established consensus on this potential connection. A prior review explored the relationship between alterations in intestinal microbial communities and their potential influence on KD development, encompassing studies available in the PubMed database up to January 2019^44^. Our review will first provide an overview of KD vasculitis and alterations of the gut microbiota, discuss the GI symptoms associated with KD, and then survey the existing studies, including studies published after 2019, performed on cohorts of KD patients as well as experimental data generated from animal models of KD. Furthermore, we discuss potential mechanisms involved in this “gut-vascular” axis in the context of KD. Finally, we will examine the potential of therapeutically targeting the microbiota in the context of KD and provide practical goals for future studies.
While the causative KD agent(s) remains unidentified, an infectious trigger(s), possibly entering through the mucosal surfaces, is suspected of initiating the inflammatory process^13,14^. Viral respiratory or intestinal infections are frequently associated with GI symptoms and alterations in the composition and function of the intestinal microbiota^45,46^. Fever and GI symptoms, such as abdominal pain, vomiting, and diarrhea, are commonly observed at the initiation of KD and precede the apparition of the main KD-associated clinical features (Figure 1A)^10,21^. Intestinal pseudo-obstruction, gallbladder hydrops, jaundice, and appendicular involvement are also reported during KD^21^ (Figure 1B). The presence of GI involvement often delays KD diagnosis and treatment, which increases the risks of a more severe outcome and the development of CA aneurysms^10,21^. Indeed, a retrospective multicenter study of a cohort of Italian KD patients presenting with (n=106) or without (n=196) GI symptoms indicates that abdominal involvement at KD onset was more frequently observed in younger patients, associated with delayed treatment and higher risk of CA aneurysm development and IVIG-resistance^22^.
Additionally, immunohistological analysis of jejunal biopsies collected from patients with KD indicates an increased presence of activated CD4^+^ T cells (HLA-DR^+^CD3^+^CD4^+^) in the lamina propria during the acute phase of the disease when compared with jejunal biopsies from healthy children or children with cow’s milk intolerance^23^ (Figure 1B). These cellular changes return to normal in the convalescent phase of the disease, one month after the fever is resolved^23^. The presence of these GI symptoms suggests a possible dysfunctional intestinal barrier, and additional indicators of intestinal barrier dysfunction in KD patients have been reported by several studies, such as higher serum levels of anti-lipid A IgA^47^, IgA anti-cardiolipin antibodies^48^, and secretory IgA (sIgA)^49,50^ (Figure 1B). sIgA is mainly produced in the intestinal mucosa, where it exerts immunoprotective functions by excluding microorganisms from the mucosal barriers^51^. Furthermore, IgA^+^ plasma cells infiltrate vascular and non-vascular tissues of KD patients^52–54^. Levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IFNγ, are elevated in patients with KD^55–60^ and can disrupt the expression of intestinal tight junctions and promote intestinal permeability^61,62^. The GI tract is home to trillions of microorganisms that influence intestinal barrier integrity and function by regulating the expression and distribution of tight junctions, supporting the biosynthesis and degradation of mucus components^3^. Alterations in microbiota composition have been linked to the development of autoimmune and immune-mediated diseases^4^. Thus, the microbiota profiles of KD patients have been a subject of great interest to the field.
The human microbiota is a highly complex and dynamic community of microorganisms that colonizes every barrier surface of the body in contact with the external environment^1–3^. The GI tract harbors trillions of bacteria from five major bacterial phyla (Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia). These symbiotic bacteria process indigestible dietary compounds, environmental toxins, and xenobiotics, protect against opportunistic pathogens, regulate host metabolism, and play a crucial role in the development and function of the immune system^30^. Gut microbes exert these effects by activating host pattern recognition receptors and releasing metabolites, which can enter circulation and reach distant tissues or act on the microbiota, influencing its overall composition.
Perturbations of the intestinal microbiota composition, or dysbiosis, disrupt microbiota-host symbiotic interactions, resulting in dysregulated host metabolism, altered immune responses, and intestinal barrier functions. Shifts in microbial structure impact the GI tract and distant organs, and have been associated with the pathogenesis of numerous diseases. Indeed, significant alterations in the structure of intestinal microbial communities have been reported in patients and experimental mouse models of inflammatory bowel diseases and non-GI diseases, such as autism, CVDs, and cardiometabolic disorders^30,63–65^. CVDs are often linked with reduced microbiota diversity, decreased abundance of short-chain fatty acids (SCFA)-producing bacteria, expansion of opportunistic pathogens, and systemic inflammation (Figure 2). SCFAs (acetate, butyrate, and propionate) are produced in the colon by bacterial fermentation of dietary carbohydrates and fibers^66^. These SCFAs promote intestinal barrier functions and regulate the host immune response. They activate various G protein-coupled receptors, such as GPR41, GPR43, and GPR109A, and inhibit histone deacetylases (HDACs)^67,68^. In mice, SCFA supplementation—whether individually or in combination—has been shown to lower blood pressure^69^, regulate the immune response after myocardial infarction^70^, and reduce the development of atherosclerosis^71^. Colonization of atherosclerotic mice with Roseburia intestinalis, which produces butyrate, reduces atherogenesis and endotoxemia by enhancing intestinal barrier function and dampening inflammation^72^. Roseburia intestinalis, as well as butyrate, reduce the growth of abdominal aortic aneurysms in mice by inhibiting neutrophil infiltration and preventing the synthetic phenotypic switch of vascular smooth muscle cells^8^. Comparable findings connecting decreased abundance of SCFA-producing bacteria were observed in patients with CVDs (Figure 2). For example, the loss of butyrate producers, particularly Roseburia, Faecalibacterium, Coprococcus and Ruminococcaceae, and the blooming of pathogens Escherichia, Streptococcus, Shigella, Klebsiella, Parabacteroides have been reported in individuals with hypertension^73–75^. Compared with healthy controls, the stool samples from patients with coronary artery disease exhibit a reduced abundance of butyrate producers Faecalibacterium, Roseburia, and Eubacterium rectale and enrichment in the opportunistic pathobionts Escherichia-Shigella, Ruminococcus gnavus and Enterococcus^76,77^. In symptomatic atherosclerotic patients, the abundance of SCFA producers Roseburia and Eubacterium is also reported as decreased, while Collinsella, Enterobacteriaceae, and Streptococcus spp. are enriched^78,79^. Patients with abdominal aortic aneurysms also exhibit intestinal dysbiosis, characterized by decreased relative abundance of the SCFA-producing bacteria Roseburia intestinalis and increased relative abundances of pathogens, such as Klebsiella quasipneumoniae, Klebsiella oxytoca, and Enterobacter asburiae^8^. These results from preclinical animal models and observational studies on cohorts of patients with cardiovascular conditions provide compelling evidence showing how the gut microbiota and its derived metabolites, particularly SCFAs, impact the development of CVDs.
Antibiotics are essential in combating infectious diseases, but they can also disrupt the microbiota’s composition and functions, leading to lasting harmful effects on the host. Indeed, although used to target pathogenic bacteria, antibiotics also impact beneficial bacteria^29^, which can lead to long-term perturbations and the disappearance of some microbial community members^80,81^. In healthy adults, bacterial richness recovers within two months of antibiotic treatment^82^. However, alterations in taxonomy, resistome, and metabolic activity may persist longer^82^. Interestingly, in an age- and sex-matched case-control retrospective study on 50 patients with KD and 200 control subjects in Japan, Fukazawa et al. reported an association between previous antibiotic exposure and KD onset^83^. In that study, the median interval between the final dose of antibiotics and the onset of KD was 2.5 months, suggesting that prior antibiotic use may contribute to the development of KD by affecting the intestinal microbiota in infants and young children^83^. Similarly, a recent retrospective case-control study with 17,818 KD patients and 89,090 matched-control subjects revealed that antibiotic usage within the past 6 or 12 months may be associated with the development of KD among children, and this correlation was most pronounced in children who received 3 or more types of antibiotics within 12 months of developing acute KD^84^
Because acute KD’s clinical and laboratory findings overlap with those of many infectious diseases, KD patients are often treated with antibiotics before diagnosis^13,85–88^. This treatment is ineffective for KD, and little is known about whether antibiotic exposure during the acute phase of the disease impacts KD’s pathophysiology and outcome. A study performed on a Canadian cohort of children with KD indicates that several clinical and laboratory features (baseline temperature, lymphocyte and monocyte counts, CRP levels) differ between KD patients responding to IVIG treatment and those who do not^87^. Among these, children with KD who were completely non-responsive to IVIG treatment were more likely to have received antibiotics during the acute phase of the disease^87^. Furthermore, two additional studies reported that antibiotic use during acute KD affected responsiveness to IVIG^89,90^. A recent nationwide epidemiologic survey conducted in Japan from 2017 to 2018 involving 28,265 KD patients revealed that prior antibiotic use in the week before the diagnosis of acute KD significantly increased the risk of development of CA lesions^28^. These studies strongly support a link between both prior antibiotic use and KD susceptibility, and antibiotic use during the acute phase of the disease and KD treatment responsiveness. However, large prospective investigations are still needed to establish a potential link between antibiotic exposure before KD diagnosis and clinical outcomes, including the risk of developing CA lesions.
The potential connection between alterations in gut microbiota composition and the development of KD in children has been investigated with culture-based techniques, 16S rRNA gene sequencing, and metagenomics approaches (Table 1 and Figure 1B)^31–43^. While these studies did not identify a consistent and reproducible microbiota signature associated with KD, they frequently report a decreased relative abundance of SCFA-producing bacteria in KD patients. Initial studies used culture-based methods to characterize the bacteria composition of jejunal biopsies^31,34^, fecal samples^32^, and throat swabs^33^. There were no differences in oral microbiota profiles between KD patients and febrile controls^33^. Still, analysis of jejunal biopsies identified several Staphylococcus and Streptococcus species only present in KD patients^31,34^. Furthermore, gram-negative bacteria isolated from the jejunal biopsies of KD patients produced heat-shock protein (hsp) 60 or exhibited superantigenic (SAg) properties, capable of impacting host immune responses^34^. Reduced levels of Lactobacillus during the acute phase of the disease were also reported in the feces of KD patients^32^. Lactobacilli support intestinal barrier functions by promoting mucus secretion, the expression of intestinal tight junctions, and the secretion of several bacterial-derived products, including SCFAs^91^. Notably, more than 50% of the KD patients in this study were treated with antibiotics before fecal sample collection, whereas none of the non-KD disease control group patients received a similar treatment^32^. Therefore, it is difficult to ascertain whether these changes are specific to KD or a consequence of the antibiotic treatment.
Kaneko et al. proposed that gut microbiota dysbiosis during KD and reduction of butyrate-producing bacteria could contribute to the development of KD pathogenesis by disrupting the balance of Th17/regulatory T (Treg) cells and potentially triggering hypercytokinemia^92^. In a preliminary study, they measured the levels of fecal SCFAs of a small cohort of healthy control children and KD patients (n=4/group) and reported a significant reduction in butyrate during acute KD^92^. However, the results need to be confirmed and reproduced in independent cohorts of KD patients due to the small sample size and the absence of a febrile control group. Kinumaki et al. performed a longitudinal metagenomic analysis of feces from 28 KD patients collected at three different time during the acute phase (patient hospital admission), the convalescent phase (patient discharged from the hospital), and 4 to 6 months post-KD diagnosis^35^. This analysis indicated an increased abundance of Streptococcus species during the acute phase of the disease. Furthermore, KD patients exhibited increased abundances of Ruminococcus, Blautia, Roseburia, and Faecalibacterium, all SCFAs producers, in fecal samples collected 4 to 6 months post-disease onset^35^. Still, most of the KD patients received antibiotic treatment during the acute phase of the disease, complicating the ability to determine whether the observed alterations in microbiota composition are a consequence of the disease or antibiotic-induced dysbiosis^35^.
Chen et al. addressed this limitation by using 16S rRNA gene sequencing to longitudinally profile the fecal microbiota communities in KD patients who did not receive antibiotics in the three months preceding diagnosis^36^. Several alterations in the intestinal microbiota’s composition during the disease’s different phases were observed^36^. Indeed, compared to healthy controls, KD patients showed an increased relative abundance of Enterococcus, Acinetobacter, Helicobacter, Lactococcus, Staphylococcus, and Butyricimonas^36^. At the same time, beneficial bacteria producing SCFAs (Prevotella, Dialister, Clostridium, Eubacterium, Roseburia, and Megasphaera) were decreased in children with KD during the acute phase of the disease compared with healthy controls^36^. In contrast, SCFA-producing bacteria Blautia, Prevotella, Dialister, Clostridium, Roseburia, Anaerostipes, Ruminococcus, and Dorea were enriched during the convalescent phase of the disease (up to 6 months post-KD)^36^. Comparable results were reported in another cohort of KD patients, with a significant decrease in the abundance of SCFA-producing Bacteroidetes and Dorea in KD children compared to healthy control patients^37^. However, neither of these studies directly assessed the impact of such alterations on serum and/or fecal SCFAs levels, or included a control group consisting of age-matched febrile patients.
In a more recent metagenomic analysis of fecal samples collected from healthy children and KD patients pre-and post-IVIG treatment, Han et al. reported a decreased abundance of beneficial species such as Akkermansia muciniphila (A. muciniphila), Faecalibacterium prausnitzii (F. prausnitzii), and Bacteroides thetaiotaomicron and ovatus, in KD patients pre-IVIG treatment, which returned to control levels after IVIG treatment^43^. The authors also analyzed the gut bacterial communities in the context of IVIG resistance and reported an association between IVIG resistance and reduction of Bacteroides thetaiotaomicron^43^. Unfortunately, this study provided no information regarding antibiotic use in the enrolled patients^43^. In a separate cohort, Teramoto et al. used 16S rRNA sequencing of fecal samples from children who had been diagnosed with KD in the 6 to 15 months before sample collection and showed that compared to healthy controls, those with a history of KD had a marked increase in the abundance of the pro-inflammatory bacteria Ruminococcus gnavus, and a decrease in the relative abundance of inflammation-suppressing SCFA-producing Blautia^40^. However, this study had significant limitations, including the absence of data on the gut microbiota composition during the acute phase of the disease for each KD patient and other confounding factors, such as diet and use of antibiotics in the 6 to 15 months post-KD, are not addressed, hindering the ability to reach definitive conclusions.
KD is a febrile disease, and a significant limitation of many of these microbiome studies is the lack of comparison between KD patients and non-KD patients with other febrile diseases^31,34–38,40,42,43^. In contrast, Fabi et al. investigated the gut microbiota composition of a small cohort of children with Henoch-Schonlein purpura (HSP), and KD patients, as well as age-matched febrile controls and healthy controls by 16S rRNA gene sequencing^39^. That study reported a reduction in the abundance of members of the Lachnospiraceae (Anaerostipes, Lachnospira, Blautia, and Roseburia) and Ruminococcaceae (Ruminococcus and Faecalibacterium), two bacterial families involved in SCFAs production, in all diseased patients compared to healthy controls^39^. Compared to healthy controls, the gut microbiota of KD patients exhibited decreased abundances of Blautia, Lachnospira, Roseburia, Ruminococcus, and Dialister^39^. However, compared with healthy controls, patients with non-KD febrile illnesses also exhibited decreased relative abundances of Blautia and Lachnospira, hinting that these alterations might not be specific to a disease but reflective of a febrile state or intense inflammation^39^. Mechanistically, several correlations have been established between levels of circulating pro-inflammatory cytokines and the abundance of different intestinal bacteria in KD patients^36,39^. While the abundances of Enterococcus and Helicobacter are positively correlated with circulating levels of IL-6^36^, inverse correlations were reported in KD patients between known SCFA producers Blautia and circulating levels of IL12p70, and between Butyricimonas and Coprococcus and circulating levels of TNF-α^39^.
Overall, the studies investigating alterations in gut bacterial communities in children with KD have several limitations that may restrict their interpretation . Different microorganisms require specific culture conditions and media; a large proportion cannot be cultured due to unknown growth requirements. Therefore, culture-based methods, such as those used in early studies^31–34^, are limited in the range of detectable organisms^93^. While some of the studies were longitudinal and collected samples from KD patients at different time points^35,36,38,43^, the inclusion of a control group of children with non-KD febrile disease is rare^32,33,39^. In some of these studies, the cohorts were small (n<10 patients) and may not adequately capture the extensive inter-individual variability of the human gut microbiota, potentially masking essential associations^38,42^. Lastly, some studies either did not consider whether patients were treated with antibiotics pre-diagnosis^39,43^ or, if noted, did not account for this factor in their analysis^32,33,35,38,40^. Since antibiotics significantly alter the microbiota composition, the reported results are challenging to interpret.
Furthermore, little is known about the functional impact of these alterations on host physiology and KD pathogenesis. The mechanisms linking alterations in gut microbiota composition to KD pathogenesis could be investigated further using experimental murine models of KD vasculitis (reviewed in^14^). Among these, the most commonly used are the Candida albicans water-soluble (CAWS) fraction and the Lactobacillus casei cell wall extract (LCWE) murine models of KD vasculitis. When injected into mice, CAWS and LCWE induce an IL-1β-dependent immune-mediated heart inflammation with histopathological features similar to those observed in heart tissues of autopsied KD patients^14,94–101^. Preliminary studies using the CAWS^102^ and the LCWE-induced^103^ models indicate alterations in the intestinal microbiota composition that functionally contribute to the development of experimental KD vasculitis. Both models show reduced abundance of several different SCFA-producers, and the studies suggest that oral supplementation of either SCFAs or bacteria known to be SCFA-producers minimizes the severity of vascular inflammation in mice^102,103^. These results suggest an indirect functional connection between the microbiome and KD pathogenesis and highlight the need for more comprehensive studies investigating gut microbiota and metabolite profiles simultaneously in KD patients.
Multiple lines of evidence suggest that the microbiota and its derived products play a role in KD; however, published studies have notable limitations, highlighting the need for larger, rigorously designed prospective investigations (Table 1 and Figure 1B). While no specific pathogenic bacteria have been reproducibly identified across the different cohorts of patients, the presence of several pathobionts was observed, and a decreased abundance of SCFA producers is frequently reported, which may persist in the long-term convalescent phase of the disease. Therefore, microbiota-targeted therapies might be useful to decrease the severity of KD inflammation by eliminating pathogenic bacteria and restoring specific symbiotic bacterial strains, such as SCFA-producing bacteria, which are associated with healthy host-microbiota homeostasis^104,105^. Such therapies could involve administering prebiotics and/or probiotics. Prebiotics are oral dietary supplements, such as fibers, that act as substrates to promote the growth of beneficial bacteria and the production of metabolites such as SCFAs, thereby regulating intestinal immune responses and barrier functions. Indeed, a phase II, randomized, placebo-controlled, double-blind clinical trial demonstrated that a prebiotic diet enriched in acetylated and butyrylated starch significantly reduced blood pressure in hypertensive patients^106^. This effect was attributed to the expansion of SCFA-producing bacteria in the gut microbiota and the increased levels of circulating acetate and butyrate^106^. Hence, similar strategies may be applicable to any inflammatory disease linked to reduced levels of SCFAs and decreased abundance of SCFA-producing bacteria, including KD.
Probiotics are live microorganisms that, when administered orally in the appropriate quantities, confer beneficial effects on the host by blocking the proliferation of pathogenic bacteria, strengthening the microbiota community structure and barrier functions. The majority of commercial probiotics include bacteria from the Lactobacillus and Bifidobacterium genera. However, F. prausnitzii and A. muciniphila are also interesting agents and are considered the next generation of probiotics due to their beneficial anti-inflammatory and metabolism-regulating effects^107,108^. Oral supplementation with either live A. muciniphila or F. prausnitzii preventively or therapeutically decreases the severity of LCWE-induced KD vasculitis in mice^103^. Similarly, oral administration of either pasteurized A. muciniphila or Amuc_1100, a protein isolated from the outer membrane of A. muciniphila, also decreases the severity of LCWE-induced KD vasculitis^103^. Mechanistically, A. muciniphila increased the thickness of the intestinal mucus layer, improved the expression of intestinal tight junction barriers, and decreased systemic immune responses^103^, in line with findings in other models^109–113^. In a randomized, double-blind- placebo-controlled pilot study, Depommier et al. demonstrated the safety and beneficial effects of oral supplementation with pasteurized A. muciniphila on volunteers with metabolic syndrome, characterized by improved insulin sensitivity and decreased insulinemia and plasma cholesterol levels^114^. Altogether, these observations should enthusiastically encourage further preclinical studies targeting the gut microbiota in the context of KD vasculitis.
Our understanding of the role of the gut microbiota in KD has advanced significantly in recent years. However, despite the increasing interest in exploring potential connections between alterations in gut microbiota composition and KD, there is a lack of direct evidence demonstrating the gut microbiota’s active involvement and functional contribution to this disease. A combination of animal, human observational and interventional studies will be necessary to determine whether and how the gut microbiota contributes to KD and to characterize the mechanisms involved. To gain more definitive insights into the role of the gut microbiota in KD, future studies should integrate both metagenomics (fecal) and metabolomics (fecal and plasma) analysis of samples from longitudinal cohorts of KD patients. Since KD is an immune-mediated vasculitis, this approach should be associated with the immune phenotyping of KD and control patients investigated, as well as correlation analysis between changes in microbiota composition, immune and clinical parameters. This combined approach will enable the detailed characterization of microbial diversity and its functional potential at the species level and the identification of bacterial-derived products, offering a deeper understanding of the microbiome’s functional capabilities during KD. Published studies aiming to characterize changes in the microbiota composition of KD patients have several limitations, such as small human cohorts, the inclusion of KD patients treated with antibiotics in the study cohorts, and/or the lack of a febrile non-KD patient control group, making it difficult to differentiate a disease-specific effect on the microbiota composition from alterations resulting from unaccounted confounding factors. Therefore, future study designs must consider these limitations carefully to minimize bias and ensure robust, accurate, and reliable outcomes. Several important questions still remain. It is still unknown whether the reported changes in bacterial community structures during acute KD contribute to disease development and pathogenesis or are a consequence of KD onset and its related inflammatory response. Current research is mostly associative and suffers from a lack of functional studies. Developing a human microbiota-associated murine model involving the transfer of fecal microbiota from KD patients into germ-free mice to test the contribution of the dysbiotic microbiota to KD vasculitis, associated with a critical and rigorous scientific approach might provide further insights into the functional extent of the gut microbiota alterations reported specifically in KD patients.
Another potential future direction of research may include the study of the role of the microbiota in conditions associated with increased development of CA lesions, such as IVIG resistance and age less than 6 months^115–117^. A retrospective study of an Italian cohort of KD patients also indicated that younger patients (< 6 months) with abdominal and GI symptoms are at the highest risk for developing CA aneurysms^22^. We hypothesize that the gut microbiota may also contribute to this increased risk of CA lesions seen in this young age group since early-life intestinal microbiota exhibit less stability, diversity, and functional complexity and stabilize into a more mature conformation within the first years of life^118^. Another research area that warrants further studies is the use of antibiotics prior to diagnosis of KD, which we hypothesize may decrease the relative abundance of SCFA-producing beneficial bacteria and lead to increased IVIG resistance and development of CA aneurysms. Indeed, a recently published large national survey from Japan recently provided data that the use of antibiotics prior to KD diagnosis may be associated with an increased risk of developing CA lesions, potentially by altering the abundance of beneficial intestinal bacteria^28^.
Studies performed on murine models and human cohorts have highlighted the therapeutic potential of targeting the gut microbiota and increasing SCFA levels to modulate a range of inflammatory disorders, including metabolic and cardiovascular diseases^65,119^. Since KD is an immune-mediated condition characterized by elevated circulating levels of pro-inflammatory cytokines and GI manifestations, and the analysis of the gut microbiota from KD patients indicates the decreased abundances of SCFA-producing bacteria, interventional approaches aiming to modulate the gut microbiota through diet, such as the supplementation of fibers or the use of probiotics, to increase the production of SCFAs and promote intestinal barrier functions, should be considered an important avenue for future research in relation to KD. In summary, the combination of multi-omics approaches characterizing the gut microbiota composition and its derived products, associated with immune phenotyping of age-matched samples from KD patients, healthy and febrile controls, with, when possible, follow-up functional studies in a human microbiota-associated murine model of KD vasculitis and prebiotics and probiotics interventional strategies will be necessary to further characterize the contribution of the gut microbiota to KD.