SEX-SPECIFIC GUT MICROBIOTA DIFFERENCES IN POST-INFLAMMATORY VISCERAL PAIN

Background: Diet is known to alter the risk of depression. Increasing data also demonstrate a causal role of the gut microbiome in mental illness, via the gut-brain axis. However, it remains unclear how diet and the microbiome mechanistically influence depression risk in humans. Leveraging dietary, metabolomics, microbiome, and depression data, we assessed how gut microbial species and their pathways may mediate the association between depression and citrus, a food group that possibly protects against risk of depression.

Methods: We conducted a prospective study in the Nurses’ Health Study II (NHSII) between 2003 and 2017 among 32,427 middle-aged women free of depression at baseline. Citrus intake was determined using validated food frequency questionnaires collected every 4 years. Depression was defined according to physician-diagnosis and antidepressant use. Between 2013-2014, 207 NHSII participants enrolled in a nested substudy, providing up to 4 stool samples (profiled by shotgun metagenomics) and a blood sample (profiled by LC-MS-based metabolomics). Cox proportional hazard models were used to relate citrus intake with depression risk. Linear mixed effects models were used to relate diet with gut microbial features, and microbial features with depression. We also associated microbial features with a depression-risk score, derived according to levels of circulating serotonin and GABA. All models were adjusted for multiple dietary, medication and lifestyle variables including age, BMI, calorie/alcohol intake, and diet quality. We validated our findings in a subcohort of 307 men in the Health Professionals Follow-up Study (HPFS).

Results: Total citrus intake was associated with a lower risk of incident depression (p_trend 0.001), with a multivariable relative risk of 0.80 (95% CI, 0.68-0.93), comparing extreme quintiles. Within the NHSII substudy, greater citrus intake was associated with increased abundance of Faecalibacterium prausnitzii (β 0.026, FDR 0.17). In turn, levels of F. prausnitzii were higher in non-depressed individuals compared to depressed participants (p 0.003). Greater abundance of F. prausnitzii was also associated with our metabolomics-based depression-risk score in the NHSII (p 0.03), and in the HPFS validation study (p 0.02). In an exploratory analysis of gut microbial pathways, S-Adenosyl-L-Methionine (SAM) cycle I, encoded by F. prausnitzii, was reduced in depressed participants.

Conclusion: Greater citrus intake was prospectively associated with lower risk of depression, and with greater abundance of F. prausnitzii. In turn, participants with depression had lower levels of F. prausnitzii and lower abundance of its genes capable of producing SAM, a compound known to have antidepressant properties. These data offer a potential mechanism by which diet influences the gut microbiome to reduce risk of depression.

Background: Experiences of discrimination are associated with adverse health outcomes, including obesity and obesity-related comorbidities. However, the underlying whole-body mechanisms remain unclear. We investigated the impact of discrimination experiences on brain reactivity to food images and associated dysregulations in the brain-gut microbiome (BGM) system. Methods: Brain responses to healthy and unhealthy food cues were obtained by magnetic resonance imaging in 107 male and female participants. Fecal samples were collected to measure fecal metabolites. The Everyday Discrimination Scale was administered to assess perceived experiences of chronic and routine unfair treatment. Structural equation modeling (SEM) was used to evaluate complex relationships between discrimination and altered bidirectional communication within the BGM system in the context of reactivity to food stimuli. Results: Discrimination was associated with increased food-cue reactivity in frontal-striatal regions involved in reward processing, motivation, and executive control, especially for unhealthy foods, as well as altered glutamate-pathway metabolites involved in oxidative stress and inflammation. SEM analyses demonstrated a significant association between discrimination-related brain and gut signatures skewed towards unhealthy foods, after adjusting for key variables such as age, sex, diet, BMI, race and socioeconomic status. Conclusions: Discrimination, as a stressor, may contribute to enhanced food-cue reactivity and BGM system disruptions that can promote unhealthy eating behaviors, leading to increased risk for obesity and related diseases. Treatments that normalize these alterations may benefit individuals with discrimination-related stress exposure.

Maternal immune activation (MIA) derived from late gestational infection such as seen in chorioamnionitis poses a significantly increased risk for neurodevelopmental deficits in the offspring. Manipulating early microbiota through maternal probiotics supplementation has been shown to be an effective means to improve outcomes, however, the mechanisms remain unclear. In this study we modeled MIA by maternal lipopolysaccharide (LPS) exposure (i.p. injection at E16) and tested the hypothesis that maternal probiotic administration during lactation may rescue the adverse neurodevelopmental outcome of MIA on offspring through the impact on blood-brain barrier (BBB) development and function. T2W, Time of flight, and T1W MRI with contrast were used to evaluate BBB development and permeability at early postnatal age (two weeks of age). Morris water maze test was used to evaluate long term cognitive function at 12 weeks of age. The effects of MIA with or without maternal Limosilactobacillus reuteri (L. reuteri) exposure on gut microbiome and systemic and brain metabolites were investigated using 16S DNA sequencing of fecal samples and metabonomic analysis at both two and 12 weeks of age. We demonstrated that MIA modeled by exposing pregnant dams to LPS induced an underdevelopment of the blood vessels, an increase in permeability and astrogliosis of BBB at pre-wean age. The BBB developmental and functional deficits early in life impaired spatial learning later in life. Maternal L. reuteri supplementation starting at birth rescued the BBB underdevelopment and dysfunction-associated cognitive function. β-diversity was significantly affected by LPS with or without maternal L. reuteri exposure. We further identified two lipid metabolites that can cross the BBB upon LPS insult and fourteen metabolic products of lipid/bile acid metabolism associated with maternal L. reuteri exposure during lactation that can cross the BBB. We conclude that maternal L. reuteri-mediated alterations in β-diversity of the microbial community and metabolic responses in the offspring provide mechanisms and potential targets for promoting BBB integrity and long-term neurodevelopmental outcomes.

Introduction. Several evidence highlights the relevance of microbiota gut-brain axis (MGB) in brain diseases, including Alzheimer's disease (AD). Indeed, AD patients display gut dysbiosis, altered intestinal barrier and enteric neurogenic/inflammatory responses that, besides bowel symptoms, contribute to brain pathology. In this context, the modulation of gut microbiota is emerging as a suitable additional therapeutical option, to target MGB, and to halt or slow down central pathology. This study examined the effects of LACTIPLANTIBACILLUS PLANTARUM (HEAL9), in a spontaneous model of AD.
Methods. Senescence-accelerated mouse prone 8 (SAMP8) mice (4 months old) were employed as a model that develops spontaneously AD and SAMR1 mice as controls. Mice were treated orally with HEAL9 1X10⁹ CFU/mouse/day or placebo for two months (n=6/group) to evaluate the effects of probiotic during the early stages of AD before the full development of brain pathology. Mice underwent Morris water maze test. Then, after assessment of fecal output over one hour, animals were sacrificed, and colonic longitudinal muscle preparations were set up in organ baths and connected to isometric transducers. Neurogenic motor responses were evoked by electrical stimulation (10 Hz) in standard Krebs solution or containing guanethidine (10 μM), L-NAME (100 μM) and NK1, NK2 and NK3 receptor antagonists (10 μM) to record cholinergic contractions. Carbachol (1 μM)-induced myogenic contractions in the presence of tetrodotoxin (1 μM) were also recorded. Plasma and brain and colonic tissues were excised and processed for the evaluation of: 1) β-amyloid (Aβ)1-42 and interleukin-(IL)-1β levels (ELISA); 2) gut barrier alterations (immunofluorescence of claudin-1 tight junction, lipopolysaccharide-binding protein (LBP) levels by ELISA).
Results. SAMP8 mice displayed: 1) cognitive dysfunctions; 2) impaired colonic transit and in vitro contractility, characterized by an altered cholinergic neurotransmission; 3) increased colonic and brain Aβ1-42, IL-1β and plasma LBP levels; 4) altered distribution and decreased expression of colonic claudin-1; 5) altered mucus layer. Intake of HEAL9 counteracted cognitive impairment, colonic motor dysfunctions and decreased brain and colonic Aβ1-42 and IL-1β and plasma LBP levels. HEAL9 also restored colonic claudin-1 expression and distribution.
Conclusions. HEAL9 exerts beneficial effects on AD, by counteracting central and enteric AD-related protein deposition and inflammation and preserving gut barrier integrity. HEAL9 also restored colonic motility through the recovery of cholinergic excitatory neurogenic motility. These results substantiate the concept that the modulation of gut microbiota can represent a suitable additional therapeutical strategy, to target the MGB and to alleviate AD and related intestinal symptoms.

Post-traumatic stress disorder (PTSD) is a mental health disorder triggered by traumatic event/s causing psychiatric and medical morbidities, as well as psychosocial distress. The neurotransmitter serotonin (5-HT) is well-recognized for regulating behavior and emotion. Dysregulation is associated with depression, anxiety, aggression, impulsivity, and suicidal behaviors, all frequently seen in PTSD. 5-HT is synthesized by tryptophan hydroxylase (TPH), acts on 5-HT receptors, and subsequently is deactivated by internalization of 5-HT via the serotonin transporter (SLC6A4, SERT). SSRIs, which target SERT, are commonly used for mood and anxiety disorders, and are the only FDA approved drugs for PTSD. However, the response rate is variable depending upon sex, genetic factors, and environmental factors. 5-HT is abundantly present and synthesized in the gut and alters microbial composition. However, the contribution of intestinal 5-HT and SERT in the progression of PTSD is not understood. Here, we investigated the role of intestinal SERT and its effect on the gut microbiota in the progression of PTSD in a sex-dependent manner. Methods: SERT^fl/flxVillin-Cre^ERT2mice (M/F, 8-12 wks) were injected with tamoxifen to induce SERT overexpression in only intestinal epithelial cells (SERT O/E^IEC), or corn oil (WT). PTSD mouse model of protracted social isolation (SI) was used as a chronic stressor to elicit aggression in resident-intruder assays. Aggression was measured by the number of attacks, duration of attacks, and onset of attacks. Intestinal mucosa, cecal contents, and brain were collected. Results showed an important role of IEC specific SERT in differentially modulating PTSD-like behavior (aggression) in males and females as evidenced by an increased aggression in isolated SERT O/E^IEC males and decreased aggression in SERT O/E^IEC single-housed females vs. their respective controls. Reduction in aggression in SERT O/E^IEC SI females was concomitant with i) an increase in BDNF and 5a reductase mRNA in amygdala & hippocampus; ii) an increase in ileal MCT10 (SLC16A10, Trp/Phe transporter); iii) increase in Tph-1, a known target of microbiota; iv) an increase in antimicrobial peptides, while isolatedSERT O/E^IEC males showed decreases in these pathways. To fully understand the mechanisms underlying these differential responses, we analyzed gut microbiota composition. Pearson correlation analysis in WT and SERT O/E^IEC(M/F) showed that gut microbial species from the order-Bacteroidales, Gastranaerophilales, and genus-Anaerotruncus positively correlated with aggression and Desulfovibrio was negatively correlated. Conclusion: These novel data suggest that aggression due to social isolation is modulated by the gut microbiota in a sex-dependent manner via mechanisms involving intestinal serotonergic machinery.

Background:
Intestinal microbial dysbiosis is now recognized as a hallmark of Parkinson’s disease (PD). Our group and others have shown that PD dysbiosis is characterized by increased pro-inflammatory bacteria and reduced abundance of short-chain fatty acid (SCFA)-producing bacteria. SCFAs stimulate enteroendocrine L-cells to secrete glucagon-like peptide-1 (GLP-1), an incretin hormone with neuroprotective properties and protective effects on the intestinal barrier. Here we hypothesize that PD dysbiosis is associated with decreased SCFAs, decreased L-cells, reduced GLP-1 secretion, and intestinal barrier dysfunction, findings that may be reversed by SCFA-promoting prebiotic therapy.

Methods:
We assembled a cohort of 19 PD patients and their spousal controls who provided stool and serum from which we measured SCFA (butyrate, propionate, and acetate) levels by GC-MS. Serum GLP-1 (45 min after consumption of a standard meal), fecal calprotectin, and serum zonulin (marker of intestinal barrier integrity) were measured by ELISA. Outcomes were reassessed after consumption of 10g of prebiotic fiber designed to increase SCFA-producing gut bacteria twice daily for 10 days and analyzed by Wilcoxon-signed rank test for pairwise comparisons. To determine L-cell density from patient-specific crypts, apical-out sigmoid colonoids and ileal enteroids were derived from 14 PD and control subjects, embedded in extracellular protein matrix, and treated with growth factors to expand Lgr5⁺ stem cells; L-cells were identified immunohistochemically by antibodies to chromogranin and GLP-1.

Results:
Here we demonstrate that compared to healthy control subjects, PD patients contain decreased SCFAs, particularly butyrate, in stool and plasma (p<0.01), corresponding to significantly decreased abundance of intestinal SCFA-producing bacteria such as Bifidobacterium as well as a 2-fold reduction in postprandial GLP-1 secretion (p=0.02) that we previously noted. PD-derived colonic and ileal organoids produce fewer L-cells than healthy organoids (trend). Furthermore, we show significantly increased calprotectin (p=0.044) and decreased intestinal barrier integrity (p<0.001) in PD patients. Our preliminary data show that prebiotic supplementation may restore plasma SCFAs, reduce fecal calprotectin, and improve intestinal barrier integrity.

Conclusions:
Intestinal dysbiosis in PD is associated with decreased 1) abundance of SCFA-producing bacteria, 2) serum and stool SCFAs, 3) GLP-1 secretion, and 4) intestinal barrier integrity, as well as increased fecal calprotectin. Preliminary data suggest these effects can be mitigated by prebiotic supplementation. These findings may hold important implications for impaired neuroprotection (GLP-1) and gut inflammation (barrier integrity and calprotectin) in PD. Future studies will determine the effects of prebiotics on clinical outcomes.

Introduction
Despite achieving endoscopic remission, up to 50% of Inflammatory Bowel Disease (IBD) patients continue to experience chronic abdominal pain, with female patients displaying an increased prevalence. The reason underlying these differences in pain perception is unknown, but the influence of the gut microbiota and sex hormones represent important potential sources for variability in pain sensitivity. Alterations in gut microbiota composition and microbial metabolites (i.e., dysbiosis) are associated with IBD pathogenesis. Previous data from our laboratory demonstrated that microbial-derived short-chain fatty acids (SCFA) were able to sensitize nociceptive neurons in a mouse model of post-inflammatory visceral pain. To date, little is known about sex differences in the gut microbiota or microbial metabolites in chronic visceral pain in IBD.

Aim
Examine the sex-specific gut microbiota differences in post-inflammatory chronic visceral pain in a mouse model of IBD.

Methods
We used the post-inflammatory DSS mouse model of chronic visceral pain. Male and cycling female mice were given 2.5% DSS in drinking water for five days and allowed to recover for 5 weeks. Visceral pain was evaluated using the visceral motor reflex (VMR) to colorectal distension five weeks after DSS treatment; pain testing in females was performed in diestrus. Fecal microbial composition was examined using 16S rRNA gene V4 region amplicon sequencing, and the metabolic profiling was examined using mass spectrometry.

Results
The severity of colitis was significantly lower in female mice than in males [Disease Activity Index at Day 12: 1.41±0.41 females, n=12; 4.41±0.31 males, n=12; p<0.001]. Despite this, post-inflammatory female mice demonstrated increased visceral hypersensitivity compared to post-inflammatory males [VMR at 60mmHg, females 0.10±0.016, n=10; males 0.07± 0.007, n=10; p=0.032] (Figure 1). Post-inflammatory females showed a significant increase (n=11; p<0.001) in alpha diversity. Beta diversity was significantly different in both post-inflammatory males (n=11; p<0.05) and females (n=11; p<0.01) when compared to baseline. An increase in differential abundance, including SCFA-producing Firmicutes like Lachnospiracea and Ruminococaceae, was seen in the post-inflammatory state in both sexes (Figure 2). Fecal acetate (n=5; p<0.0001) and butyrate (n=5; p=0.0123) were increased in post-inflammatory males compared to controls, while SCFA were not significantly different in post-inflammatory females compared to controls.

Conclusion
These data suggest that dysbiosis exists in the post-inflammatory state in both female and male mice, with significant sex differences in microbial metabolites. Identifying sex-specific differences in the gut microbiome in post-inflammatory visceral pain in IBD may allow us to define novel therapeutic approaches for chronic pain in IBD patients.