Relationships of gut microbiota, short-chain fatty acids, inflammation, and the gut barrier in Parkinson’s disease

Background Previous studies have reported that gut microbiota, permeability, short-chain fatty acids (SCFAs), and inflammation are altered in Parkinson’s disease (PD), but how these factors are linked and how they contribute to disease processes and symptoms remains uncertain. This study sought to compare and identify associations among these factors in PD patients and controls to elucidate their interrelations and links to clinical manifestations of PD. Methods Stool and plasma samples and clinical data were collected from 55 PD patients and 56 controls. Levels of stool SCFAs and stool and plasma inflammatory and permeability markers were compared between patients and controls and related to one another and to the gut microbiota. Results Calprotectin was increased and SCFAs decreased in stool in PD in a sex-dependent manner. Inflammatory markers in plasma and stool were neither intercorrelated nor strongly associated with SCFA levels. Age at PD onset was positively correlated with SCFAs and negatively correlated with CXCL8 and IL-1β in stool. Fecal zonulin correlated positively with fecal NGAL and negatively with PD motor and non-motor symptoms. Microbiota diversity and composition were linked to levels of SCFAs, inflammatory factors, and zonulin in stool. Certain relationships differed between patients and controls and by sex. Conclusions Intestinal inflammatory responses and reductions in fecal SCFAs occur in PD, are related to the microbiota and to disease onset, and are not reflected in plasma inflammatory profiles. Some of these relationships are distinct in PD and are sex-dependent. This study revealed potential alterations in microbiota-host interactions and links between earlier PD onset and intestinal inflammatory responses and reduced SCFA levels, highlighting candidate molecules and pathways which may contribute to PD pathogenesis and clinical presentation and which warrant further investigation. Supplementary Information The online version contains supplementary material available at 10.1186/s13024-021-00427-6.


Introduction
More than thirteen studies from three continents have established that the gut microbial communities of patients with Parkinson's disease (PD) differ from those of healthy subjects. [1][2][3] Most of these studies have focused on profiling the bacteria present in stool samples but have not linked these findings to functional consequences on microbiome-host interactions.
Microbially-produced short-chain fatty acids (SCFAs) and regulation of immune responses and gut permeability are some of the proposed mechanisms by which gut microbes could impact brain health and function. 4,5 SCFAs, particularly acetic, propionic, and butyric acid, are the main end product of bacterial fiber fermentation in the gut. 6 They have many effects on host physiology including acting as an energy source for colonocytes, 7,8 regulating the gut barrier, 9 and influencing inflammatory responses. 10 SCFAs have been suggested as key mediators in microbiota-gut-brain interactions. 11 In a study contrasting fecal SCFA levels in PD patients and control subjects, decreased concentrations of SCFAs, particularly butyric acid, in PD patients were linked to microbiota alterations, 12 and several bacterial taxa reportedly reduced in PD are SCFA producers. 2,3,13 Inflammation is a widely recognized hallmark of PD. Increased levels of a variety of inflammatory molecules in brain and CSF [14][15][16] as well as blood [17][18][19] from PD patients have been documented. While the same specific cytokines, chemokines, and other inflammatory markers are not consistently implicated, IL-6, TNF, IL-1β, CRP, IL-10, CCL5, and IL-2 are among the most commonly dysregulated in the peripheral blood of PD patients. 18 It has been suggested that this inflammation may result from intestinal barrier deficiency which could increase systemic exposure to inflammatory microbial products such as lipopolysaccharide (LPS), a component of bacterial cell walls. 5,20 Experiments measuring excretion of ingested sugars suggest increased permeability of the colon (but not the small intestine) in PD. 21-23 Higher levels of zonulin and alpha-1-antitrypsin, indicators of gut permeability, have been found in PD patients' stool relative to controls, 24 although this difference has not been consistently observed. 25 Additionally, increased detection of LPS 26 and decreased LPS-binding protein (LBP) 21, 27 in blood and plasma from PD patients suggest greater exposure of peripheral tissues and immune cells to LPS, supporting the existence of PD-related gut barrier dysfunction. All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06. 15.20131011 doi: medRxiv preprint In recent years, there has been increasing recognition of a low-grade inflammatory state in the gut in PD. Studies have reported increased expression of genes encoding proinflammatory cytokines and chemokines in gut tissue from PD patients compared to controls, 22,28 and higher levels of IL-1α, IL-1β, CXCL8 (also known as IL-8), and CRP have been found in stool. 29 The neutrophil-associated protein calprotectin is an indicator of gut inflammation in inflammatory bowel disease, 30 and increased levels of calprotectin have been found in the stool of PD patients relative to controls. 24,25 Finally, higher numbers of CD3+ T cells and cells expressing the LPS receptor TLR4 have been identified in colon tissue from PD patients. When TLR4 was knocked out in a mouse model of PD pathology, symptoms were mitigated. 22 Taken together, these findings support the hypothesis that intestinal bacteria and their metabolites along with inflammation and barrier dysfunction impact PD pathophysiology.
While there have been several studies evaluating inflammatory and gut permeability markers in PD, this study addresses the gap in knowledge regarding the interplay of these molecules in the blood and the gut and with the gut microbiota and SCFAs in PD patients and healthy control subjects.

Study subjects and sampling
The study was approved by the ethics committee of the Hospital District of Helsinki and Uusimaa. All participants gave informed consent. The study subjects were originally recruited for a pilot study of gut microbiota and PD. 31 Samples for the present study were collected at a follow-up timepoint together with extensive clinical data, including questionnaires on diet, nonmotor symptoms, and PD severity; full details have been published previously. 32 Subjects collected stool samples at home into tubes with DNA stabilizer for microbiota analyses and tubes without preservatives for SCFA and immunological analyses. They kept the samples refrigerated until transport to the clinic (up to 3 days) for storage at −80°C. Venous blood samples were collected into EDTA tubes, centrifuged (10 min, 3000 rpm), and plasma was divided into aliquots and stored at -80°C. Samples were distributed to the laboratories by overnight shipping on dry ice. After excluding subjects with missing values for microbiota, acetic, butyric, or propionic acid, or most immune markers, the total number of subjects remaining for analysis was 111 (55 patients, 56 control subjects). All rights reserved. No reuse allowed without permission.
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Short-chain fatty acid measurements
Detection and quantification of SCFAs in the stool samples were performed at the Norwegian University of Life Sciences using a Trace 1310 gas chromatograph (Thermo Fisher Scientific).
Full details are available in Supplementary materials. The V-PLEX kit was also used to measure inflammatory mediators in plasma, as was the Human LBP Kit (MSD K151IYC). Stool and plasma samples were prepared, assays run, and results analyzed according to manufacturers' protocols by the Emory Multiplexed Immunoassay Core. Full details are available in Supplementary materials.

Statistical analyses
All statistical analyses were performed in R 37 with packages including phyloseq, 38  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

PD patients have higher calprotectin and lower SCFA levels in stool and lower CXCL8 levels in plasma compared to controls
Patient and control groups were similar with regard to basic demographics such as age, sex, and BMI; however, as we knew from previous analyses of the same subjects, 31, 32 the groups differed regarding medications, medical history, and various symptom scores. A higher percentage of controls reported a history of stroke and use of medications for high blood pressure and cholesterol, and PD patients scored higher on scales of non-motor symptoms, gastrointestinal problems, and constipation (Table 1). Contrasting inflammatory markers and SCFAs between PD patients and control subjects, patients had lower levels of butyric and propionic acid and higher levels of calprotectin in their stool (Table S1) and lower levels of CXCL8 in plasma (Table S1). When the data were stratified by sex, the differences were particularly prominent for butyric acid in males and plasma CXCL8 and stool calprotectin in females ( Fig. 1, Table S1).

Inflammatory markers in plasma and stool are not correlated, but stool SCFAs, zonulin, and stool and plasma cytokines are related to PD onset and symptom severity
The inflammatory markers measured from the same sample material (plasma or stool) were largely intercorrelated, but we found no correlations between measurements of the same markers in stool and plasma (Fig. S1). PCAs for combining the most correlated markers resulted in one principal component (PC) for stool (summarizing IL-10, IL-12p70, IL-13, TNF, IL-6, and IFNγ), and two plasma PCs (PC1: IL-10, IL-4, TNF; PC2: IL-1β, IL-2, IL-12p70, IL-13) (Supplementary R Markdown). In stool, the inflammatory markers neutrophil gelatinaseassociated lipocalin (NGAL) and calprotectin and the gut permeability marker zonulin were significantly correlated (Fig. S1). In general, calprotectin and zonulin showed fewer correlations with other stool markers in the PD group as compared to the control group. Except for correlations with stool CXCL8 and IL-1β in the control group, none of the three most abundant SCFAs was significantly correlated with any of the markers in stool or plasma (Fig. S1).
Contrasting clinical variables against SCFAs and inflammatory markers suggested inverse correlations between stool SCFAs and several PD-related clinical variables such as the Non-All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.15.20131011 doi: medRxiv preprint Motor Symptoms Scale (NMSS) total score, the Rome III constipation/defecation subscore, and the Geriatric Depression Scale-15 (GDS15) (Figs. 2A, S2A). A similar pattern was seen for the Rome III constipation/defecation subscore, Rome III IBS criteria, and NMSS score and plasma CXCL8 (Figs. 2B, S2A). These correlations were significant for the entire cohort but not when evaluated only within PD patients. Butyric acid levels were correlated, however, with the age of onset for motor and non-motor symptoms of PD, while CXCL8 and IL-1β levels in stool were inversely correlated with the age of motor symptom onset ( Fig. 2A, 2C, S2B). In PD patients, calprotectin levels were inversely linked to IBS symptoms, and stool zonulin was inversely correlated with variables related to PD severity, including the Unified Parkinson's Disease Rating Scale (UPDRS) score, GDS15, and NMSS score (Fig. 2C, S2B). Symptom severityrelated correlations remained significant after correcting for disease duration (Supplementary R Markdown).

Microbial alpha diversity is inversely associated with stool inflammatory and permeability markers and SCFAs
Microbial alpha diversity indices, which indicate richness and evenness of bacterial taxa, were inversely correlated with total SCFAs, butyric and propionic acid, as well as with NGAL, calprotectin, zonulin, and IL-1β in stool (Fig. S3). Correlations between alpha diversity and SCFAs, NGAL, and zonulin were most evident in control subjects while correlations with calprotectin were most apparent in PD patients. In patients only, a significant inverse correlation between the Shannon diversity index and stool CXCL8 was also found (Fig. S3). Linear regression modeling corrected for sex and PD/control status suggested that the relationship between alpha diversity and stool inflammatory markers was independent of sex except for calprotectin, for which an inverse relationship was observed in females but not in males (Table   S2A-C, Fig. 3A). There was a significant interaction between PD/control status and alpha diversity for propionic acid, showing an inverse correlation between microbial alpha diversity and propionic acid concentration in controls but not in PD patients (Fig. 3B). The total SCFAs variable showed a similar trend (Table S2B). The interaction in the propionic acid model remained significant after confounder correction (Table S2D).

Microbial beta diversity is related to SCFA levels and inflammatory and permeability markers in stool
Contrasting community dissimilarity (beta diversity) and SCFAs or inflammatory markers, we found significant effects for total SCFAs, acetic, butyric, and propionic acid as well as stool All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.15.20131011 doi: medRxiv preprint NGAL, zonulin, IL2, and the stool marker PC in full data, with or without correction for PD/control status, sex, constipation, and BMI (Table S3A). The effect of calprotectin, while not statistically significant, was worth noting (p = 0.058 for full data, p = 0.079 when corrected for confounders). Control-only comparisons showed significant effects for SCFAs, NGAL, and zonulin; only butyric acid and zonulin were significant in PD-only comparisons (Table S3A, Fig.   S4). These two variables also had a significant interaction with PD/control status when tested with the variable split into two categories by median (Table S3B).

The Prevotella enterotype is associated with higher levels of butyric acid and lower levels of NGAL and zonulin in stool
One broad characterization method for microbiota composition is defining enterotypes based on certain indicator species in bacterial communities. 36 In our subjects, stool zonulin, NGAL, propionic acid, and butyric acid had differences in concentrations between enterotypes (Table   S4). Butyric acid levels were higher in PD patients with the Prevotella enterotype than the other two enterotypes, and control subjects with this enterotype had the lowest concentrations of NGAL and zonulin (Fig. S5). The findings regarding NGAL (full data and controls) and zonulin (controls only) remained significant after FDR correction (Table S4).

Specific bacterial taxa are associated with levels of stool SCFAs and inflammatory and permeability markers
Guided by the associations with beta diversity, we explored the associations of specific bacterial taxa with the three most abundant SCFAs and with stool NGAL, calprotectin, and zonulin. The abundances of Butyricicoccus, Clostridium sensu stricto, and Roseburia were positively correlated with levels of SCFAs while the abundances of Akkermansia, Escherichia/Shigella, Flavonifractor, Intestinimonas, Phascolarctobacterium, and Sporobacter decreased with increasing SCFA concentrations (Fig. 4, S6, Table S5A). Similar patterns could be seen for the parent families of these taxa, such as a positive association between butyric acid and Lachnospiraceae and a negative one for Enterobacteriaceae and Verrucomicrobiaceae (Table   S5A). A positive association between SCFAs and Bacteroides was observed only in controls, while a negative association with Bifidobacterium was observed only in PD patients (Fig. 4, S6, Table S5A).
Microbiota associations with the stool markers differed between PD patients and control subjects. Stool NGAL levels were positively associated with Bifidobacterium in PD but with All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Bacteroides and Roseburia in controls (Fig. 4, S7A, Table S5B). Relative abundance of Prevotella was negatively associated with both NGAL and zonulin in controls only (Fig. 4, S7A- Table S5B). The only microbiota-immune association found to be significant in both PD patients and controls was the negative relationship between stool zonulin and the genus Coprococcus (Fig. 4, S7B, Table S5B), a taxon known to have a strong influence on gut permeability 41 and stool zonulin levels. 42 Other associations with low-abundance taxa were statistically significant but were driven by a small number of data points, precluding confident interpretation (Fig. S7).

Discussion
Numerous studies have reported abnormalities in the gut microbiome, levels of SCFAs, peripheral immune responses, and intestinal permeability in PD; however, this study provides for the first time an integrative analysis of these factors in the same subjects.
Efforts have been made to identify biomarkers of PD in both blood and stool, but the relationship between the levels of inflammatory markers in the gut and in circulation in this disorder had not been elucidated. We determined that levels of inflammation-associated molecules in the plasma and in the stool were highly correlated within each sample type. We found essentially no correlation, however, between levels of inflammatory markers in plasma and stool. This indicates that systemic and gut immune responses are distinct and, thus, measurements from one site are unlikely to represent the nature of the responses in the other.
In the plasma, we found a significant decrease in levels of the chemokine CXCL8 (also known as IL-8) in female PD patients as well as an inverse correlation between CXCL8 and non-motor symptom severity scores in the full cohort. CXCL8 is the primary chemoattractant for neutrophils and is commonly produced in inflammatory responses to stimulate and recruit these phagocytic cells. A previous study has reported a positive correlation between CXCL8 levels in the serum and the degree of PD-related disability, 43 rendering our findings somewhat unexpected. A possible explanation is that plasma CXCL8 levels in this cohort are significantly higher in subjects with a history of stroke (Fig. 2), the majority of whom are controls.
In stool, we documented lower levels of SCFAs and higher levels of calprotectin in PD patients. This is consistent with other reports of SCFA and SCFA-producing bacteria reductions 3, 12, 13, 19, 22, 31 as well as increased stool calprotectin 24,25 in PD. While SCFAs and calprotectin are beginning to emerge as some of the most reliable indicators of a dysregulated and inflammatory All rights reserved. No reuse allowed without permission.
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Levels of butyric acid specifically were correlated with the age of onset for both motor and nonmotor PD symptoms even after accounting for disease duration, raising the possibility that higher butyric acid levels could be protective and delay disease onset. We also found inverse correlations between stool SCFA levels and non-motor, particularly gastrointestinal and depressive, symptoms. This correlation was not significant in just the PD subset of the cohort, however, which could be due to a lack of statistical power or to a difference between the control and patient groups.
Though SCFAs are known to have immunomodulatory and barrier-promoting properties, we found no significant correlations between the most abundant stool SCFAs and immune-or permeability-related factors in stool or plasma. While it is possible that SCFA levels may directly impact immunological pathways not assessed in this study, the lack of clear association between SCFA production and inflammation likely evinces the complexity of the relationship between these physiological processes. Both pro-and anti-inflammatory activities of SCFAs have been documented, 44,45 and dietary supplementation of SCFAs produces variable effects on inflammation which appear to be strongly influenced by the range of SCFA concentrations involved. 46 Further investigation of the effects of SCFAs on immune responses in the gut, the blood, and the central nervous system in the context of PD pathology is needed.
NGAL, an epithelium-derived antimicrobial glycoprotein overexpressed in inflammatory conditions, 47 has been found at higher levels in the plasma of PD patients compared to controls. 43 To the best of our knowledge, this comparison had not been performed in stool, and we found no significant differences in fecal NGAL levels between patients and controls. We also found no significant indications of greater intestinal permeability in this cohort of PD patients as measured by stool zonulin or plasma LBP, though other studies have. 21, 24, 27 On the contrary, we found that higher zonulin levels were associated with less severe clinical manifestations of PD. This could be a reflection of the diversity of motor and non-motor symptom subtypes in PD. 48 Longitudinal studies assessing intestinal permeability beginning in the earliest stages of PD would be beneficial to determine its impact on disease symptoms and progression.
It has been proposed that intestinal inflammation in PD is related to gut permeability and alterations in microbiota composition and can contribute to disease pathogenesis. 5 In this study, we found that levels of fecal NGAL were positively correlated with fecal zonulin, supporting a relationship between intestinal inflammation and permeability. Levels of inflammatory and All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted June 19, 2020. . https://doi.org/10.1101/2020.06.15.20131011 doi: medRxiv preprint permeability markers were also associated with the stool microbiota. Stool NGAL, calprotectin, zonulin, and CXCL8 decreased with increasing alpha diversity, consistent with the concept that an inflamed gut environment exerts selective pressure on the microbiota. NGAL, IL-2, the stool marker PC, and zonulin were also significantly associated with alterations in beta diversity, and the association with calprotectin nearly reached the threshold for significance. Furthermore, the inflammatory factors CXCL8 and IL-1β in stool, which were found to be increased in a different cohort of PD patients, 29 were negatively correlated with the age of PD motor symptom onset in this study, supporting the involvement of gut inflammation in the development of PD pathology.
A notable finding in this study is that the relationships between gut microbes and inflammatory factors and between microbes and their metabolites such as SCFAs in stool differ between PD patients and controls. In control subjects only, SCFAs were inversely associated with alpha diversity, and SCFA associations with beta diversity were stronger in controls than PD patients.
Conversely, levels of butyric acid in stool differed significantly by enterotype only among PD patients. Relationships between SCFAs and individual taxa were largely concordant in both subject groups, including positive correlations between stool SCFAs and well-known SCFA producers like Butyricicoccus and Roseburia and a negative correlation with Akkermansia, which SCFAs are known to inhibit. 49 On the other hand, a positive correlation between SCFAs and relative abundance of Bacteroides was found only in controls while a negative correlation with Bifidobacterium was found only in PD patients.
These discrepancies could reflect differences in microbiota composition between patients and controls; Bacteroides is more abundant in controls, and Bifidobacterium is more abundant in PD patients in this cohort, 32 which could increase their functional impacts within their respective microbial communities. This could explain the positive association between SCFAs and Bacteroides, a producer of SCFAs, in controls, but not necessarily the inverse relationship between Bifidobacterium and butyric acid in PD patients. Increases in Bifidobacterium are considered butyrogenic due to cross-feeding of butyrate-producing bacteria, 50 but the opposite association was found in our PD cohort, suggesting an altered function or strain profile of the Bifidobacterium genus in the metabolic network of the PD microbiota. Further evidence that Bifidobacterium may not be performing a homeostatic function in PD is its positive correlation with the inflammatory marker NGAL in patients only.
PD patients in this cohort had reduced abundance of Prevotella, 32 and significant links between lower levels of fecal zonulin and NGAL and Prevotella abundance and enterotype were All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020.    Confounders included in confounder-corrected models were Rome III 9-15 sum score and sex.

Tables
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.      Table S1. Marker and SCFA concentrations by PD/control status and sex All rights reserved. No reuse allowed without permission.
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All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted June 19, 2020. . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.