Fecal Microbial Transplant in Individuals With Immune-Mediated Dry Eye





Purpose: To evaluate the safety of the Fecal Microbial Transplant for Sjogren Syndrome (FMT) trial in individuals with immune-mediated dry eye (DE).


Design: Open-label, nonrandomized clinical trial.


Methods: The study population included 10 individuals with DE symptoms and signs meeting criteria for Sjögren or positive early Sjögren markers. Procedures were 2 FMTs from a single healthy donor delivered via enema, 1 week apart. The primary outcome measure was safety. In addition, gut microbiome profiles, DE metrics, and T-cell profiles in blood were examined at baseline before FMT, and at 1 week, 1 month, and 3 months after FMT.


Results: The mean age of the population was 60.4 years; 30% were male; 50% were white; and 50% were Hispanic. At baseline, all subjects had significantly different gut microbiome profiles from the donor, including higher mean diversity indices. Subjects had a decreased abundance of genera Faecalibacterium, Prevotella , and Ruminococcus and an increased abundance of genera Alistipes, Streptococcus , and Blautia compared to the donor. Effector and regulatory T-cell profiles were positively correlated with each other and with DE symptom severity (T helper 1 cells [Th 1 ]; r = .76; P = .01; Th 17 : r = 0.83; P = .003; CD25: r = 0.66; P = .04; FoxP3: r = 0.68; P = .03). No adverse events were noted with FMT. After FMT, gut microbiome profiles in 8 subjects moved closer to the donor’s profile. As a group, gut microbiome profiles at all follow-up time points were more similar to the original recipients’ than the donor’s microbiome; however, certain phyla, classes, and genera operational taxonomic unit (OTU) numbers remained closer to the donor vs recipients’ baseline profiles out to 3 months. Five individuals subjectively reported improved dry eye symptoms 3 months after FMT.


Conclusions: FMT was safely performed in individuals with immune-mediated DE, with certain bacterial profiles resembling the donor out to 3 months after FMT. One-half the subjects reported improved DE symptoms. The most effective FMT administration method has yet to be determined.


D ry eye (DE) is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film accompanied by ocular symptoms. Tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles. Many individuals with DE have associated systemic immune abnormalities, with Sjögren disease being a common comorbidity. Chronic CD4+ T-cell infiltration, autoreactive B cells, proinflammatory cytokines, and acinar cell apoptosis in the salivary and lacrimal glands have been found in individuals with Sjögren disease and are believed to drive DE and other disease manifestations. , Interestingly, many of these pathophysiologic contributors are associated with gut microbiome dysbiosis, which has been described in Sjögren disease , as well as in other autoimmune diseases. Currently, however, it is not understood whether these abnormalities are contributors to or consequences of disease.


There is biologic plausibility that gut dysbiosis may contribute to immune-mediated DE and other autoimmune diseases. It is well-established that the gut microbiome impacts systemic inflammation and immunity through the generation of proinflammatory effector T cells (eg, T helper 1 cells [Th 17 ]) and anti-inflammatory regulatory T (Treg) cells. , For example, specific Clostridia species have been shown to induce Th 17 cell proliferation in intestinal and extraintestinal sites, whereas other Clostridia species produce short chain fatty acids which support Treg development. Similarly, certain Bacteroides species express molecules that suppress Th 17 inflammatory responses. An optimal balance between pro- and anti-inflammatory T cells is important locally to protect the mucosa from pathogenic microorganisms while also limiting excessive T-cell responses. Various mediators have been implicated in maintaining this balance including transforming growth factor-beta (TGF-β), interleukin-6 (IL-6), retinoic acid, and short chain fatty acids.


Various autoimmune diseases have been associated with gut dysbiosis. For example, decreased gut microbial diversity has been found in individuals with rheumatoid arthritis (RA), with a lower abundance of common commensals such as Bifidobacteria and Bacteroides and an increase in Prevotella copri . , Importantly, beyond its presence, P copri has been shown to have immune relevance in RA. Specifically, a 27-kDa protein of P copri stimulated effector T-cell responses in 17 of 40 individuals with RA. Additionally, 41 of 127 individuals with RA had IgG or IgA antibody reactivity with P copri , yet this was rarely found in individuals with other types of arthritis. These data suggest that gut dysbiosis may contribute to disease manifestations in several immune-mediated diseases, including DE. As such, microbiome manipulation by way of dietary changes, probiotics, or fecal microbial transplant (FMT) has been suggested as potential therapies for DE.


In FMT, intestinal microbiota are transferred from a healthy donor to the patient, with the goal of replacing an abnormal gut microbiome with a stable and healthy one. FMT is remarkably effective in treating Clostridium difficile infections, leading to eradication of infection and symptoms in 90% of individuals after 1 treatment, according to a meta-analysis. FMT has also been used as a treatment for autoimmune diseases of the gastrointestinal (GI) tract. In a randomized control trial of 75 patients, weekly FMT from healthy anonymous donors for 6 weeks induced a significantly larger ulcerative colitis (UC) remission rate (24%) at week 7 compared to placebo (5%). FMT has also been applied to immune diseases outside of the GI tract, including metabolic syndrome, multiple sclerosis, and idiopathic thrombocytopenic purpura. For example, 9 individuals’ metabolic syndrome had increased insulin sensitivity 6 weeks after 1 dose of FMT delivered via gastroduodenal tube from lean donors. In another open label study, 3 individuals with multiple sclerosis and constipation underwent FMT daily for 1 to 2 weeks and noted an improvement in neurological symptoms, including regaining the ability to walk, decreased paresthesia, and improved energy levels. Finally, an individual with UC and idiopathic thrombocytopenic purpura underwent 1 session of FMT via enema with regression of UC symptoms and normalization of platelet counts, effects that lasted for years after transplantation. Most relevant to DE, FMT has also been used in graft-vs-host disease (GVHD), a condition in which DE is an important component. In 1 open label study, 4 individuals underwent FMT (1 or 2 doses, 1 week apart, via enema from healthy spouses or relatives) with subsequent increased abundances of beneficial bacteria Lactobacillus, Bacteroides, Bifidobacterium , and Faecalibacterium at 4 weeks post-FMT. Concomitantly, GI symptoms such as defecation consistency and frequency also improved. The success of FMT in the above conditions suggests its potential clinical applications in other autoimmune diseases associated with gut dysbiosis.


We previously found that individuals with immune-mediated DE, including those with Sjögren disease, had gut microbiome alterations compared to healthy controls. Specifically, individuals with DE had a more diverse phyla and different genera than controls, including a decrease in Faecalibacterium and Viellonella and increases in Megasphaera, Parabacteroides , and Prevotella . Based on our preliminary work and the use of FMT in GVHD, we hypothesized that gut microbiome modulation through FMT may be a potential therapeutic approach for immune-mediated DE.


METHODS


STUDY POPULATION


The study population included 10 individuals with DE symptoms (Dry Eye Questionnaire ≥6) and signs (corneal staining score ≥2 on the National Eye Institute scale) 28 that, first, met the 2016 American College of Rheumatology criteria for Sjögren disease, having a total weighted score of ≥4 from the sum of the following: (1) anti-SSA/Ro antibody positivity and focal lymphocytic sialadenitis with a focus score of ≥1 foci/4 mm 2 , each scoring 3; (2) an abnormal ocular staining score of ≥5, a Schirmer’s test result of ≤5 mm/5 minutes, and an unstimulated salivary flow rate of ≤0.1 mL/minute, each scoring 1 or, second, had 1 or more elevated early Sjögren markers (antibodies against salivary protein 1 [SP1], parotid secretory protein [PSP], and carbonic anhydrase 6 [CA6] at a level of 20 endotoxin units [EU]/mL or greater).


ETHICAL APPROVAL


The University of Miami Institutional Review Board approved the prospective evaluation and treatment of patients. Informed consent was obtained from all subjects, and the study was adherent with the principles of the Declaration of Helsinki. The trial was registered as Fecal Microbial Transplant (FMT) for Sjogrens Syndrome (ClinicalTrials.gov identifier NCT03926286) and was performed under Investigational New Drug (IND) number 17994 granted by the US Food and Drug Administration.


CLINICAL METRICS


Demographic information for each participant was collected including age, sex, race, ethnicity, past ocular and medical history, and current medications. A complete ophthalmic examination was performed. Patients filled out standardized questionnaires for dry eye, pain, GI symptoms, and mental health (patient health questionnaire PHQ-9).


DE SYMPTOMS


At each visit, participants completed the standardized DE symptom questionnaires, DEQ5 27 (score: 0-22), and the Ocular Surface Disease Index (OSDI) (score: 0-100).


DE SIGNS


Participants underwent a complete ocular surface examination of both eyes in the following order:



  • 1.

    Ocular surface inflammation via matrix metalloproteinase 9 (MMP9) levels (InflammaDry; Quidel), graded as absent or present based on the presence of a pink line (0 = no line; 1 = pink line present).


  • 2.

    Tear breakup time using fluorescein stain measured 3 times in each eye and averaged.


  • 3.

    Corneal staining using fluorescein graded to the National Eye Institute scale, which assesses 5 areas of the cornea on a 0 to 3 scale (total scale: 0-15).


  • 4.

    Basal tear production after anesthesia placement (measured in millimeters at 5 minutes) using Schirmer strips.


  • 5.

    Meibum quality evaluated after expression with intermediate pressure applied to the lower eyelid (0 = clear; 1 = cloudy; 2 = granular; 3 = toothpaste; 4 = no meibum extracted).



IMMUNE PROFILES


Blood was obtained at each visit and sent for flow cytometry (Duraclone IF; Beckman Coulter). T-cell profiles obtained included percentages of effector T cells (Th 1 cells defined by CD3+CD4+interferon-gamma [IFN-γ]+ and Th 17 cells, defined by CD3+CD4+interleukin-17 [IL1-7]+), and Treg cells (CD25, defined by CD3+CD4+CD25hi, and FoxP3, defined by CD3+CD4+FoxP3+).


INTERVENTION


Individuals received 2 FMT (OpenBiome) via enema 1 week apart. All FMTs came from the same healthy donor (33-year-old white female with a body mass index of 20.3) with no known ocular or medical diagnoses. Three aliquots of product (90 cm 3 total) were thawed at room temperature for at least 45 minutes prior to delivery and poured into a hanging enema bag with a lubricated tip. Enemas were delivered in a private room by a registered nurse. Individuals were placed in a left lateral decubitus position with knees bent to receive the enema over a period of ∼2 minutes and asked to hold the product in for 1 hour after delivery. Monitoring for adverse events occurred during that time. Vital signs were checked by a nurse pre- and postdelivery.


TIME POINTS OF DATA COLLECTION


Individuals underwent 4 clinical examinations, 1 prior to FMT (baseline), and then 1 week, 1 month, and 3 months after FMT. An at-home stool sample was collected at each of these time points (4 total).


PROCESSING OF STOOL SPECIMENS


Stool samples were collected and placed in a glycerol suspension, homogenized, and sent to OpenBiome. Specimens were then frozen at −80°C until analysis. Total DNA was isolated using the Power-soil/fecal DNA isolation kit (Mo-Bio) as per manufacturer’s specifications. All samples were quantified using the Qubit Quant-iT dsDNA Broad-Range Kit (Life Technologies) to ensure that they met minimum concentration and mass of DNA. To enrich the sample for the bacterial 16S V4 rDNA region, DNA was amplified using fusion primers designed against the surrounding conserved regions that are tailed with sequences to incorporate flow cell adapters and indexing barcodes (Illumina). Each sample was polymerase chain reaction amplified with 2 differently barcoded V4V5 fusion primers and were advanced for pooling and sequencing. For each sample, amplified products were concentrated using a solid-phase reversible immobilization method for the purification of polymerase chain reaction products and quantified by electrophoresis using an (Agilent) 2100 Bioanalyzer. The pooled 16S V4V5-enriched, amplified, barcoded samples were loaded into the MiSeq cartridge (Illumina) and then onto the instrument along with the flow cell. After cluster formation on the MiSeq instrument (Illumina), the amplicons were sequenced for 250 cycles with custom primers designed for paired-end sequencing.


In addition to patient samples, reagent controls were supplied in triplicate as background. Samples producing amplicons at later cycles compared to majority of samples were concentrated using Agencourt AMPureXP beads (Beckman Coulter). All samples (donor and subjects) were sequenced together after barcode normalization subsequent to a preliminary sequencing run.


Sequences were quality filtered and demultiplexed using exact matches to the supplied DNA barcodes and primers, using QIIME 2.0 software. Resulting sequences were clustered at 97% to obtain phylogenetic identities using SILVA database (version 123). OTU tables were rarefied to the sample containing the lowest number of sequences in each analysis.


STATISTICAL ANALYSIS


Descriptive statistics were first used to summarize patient demographic and clinical information.


Comparison of diversity between donor and recipients


α-Diversity matrices were calculated using QIIME version 2.0 for the donor at baseline and the recipients’ baseline profiles, and at 1 week, 1 month, and 3 months post-FMT. Mean differences between the groups (donor vs recipient) were compared using an independent Student t -test and over time within recipients using paired t -tests. Both Shannon index (which takes into account both the number of species present and the relative abundance of each species) and Chao 1 (which assumes that the number of organisms identified for a taxa has a Poisson distribution and corrects for variance) were calculated and compared. Chao 1 is especially useful for data sets skewed toward low-abundance cells, as is often the case with microbes. In both cases (Shannon and Chao1), an increase in value is correlated with increased richness and evenness of the species (OTUs). β-Diversity was compared via UNIFrac principal coordinate analysis (pCOA) using observation ID (OTU) level. The permutational multivariate analysis of variance (PERMANOVA) test was utilized to find significant whole-microbiome differences among discrete categorical or continuous variables with randomization/Monte Carlo permutation tests (with Bonferroni correction). The fraction of permutations with greater distinction among categories (larger cross-category differences) than that observed with the non-permuted data were reported as the P value. False discovery rate corrected P value ( q value) of <0.05 was considered significant across groups.


Descriptive examination of microbiome composition


Cladogram qualitative analysis was used to examine relative compositions of bacteria with respect to phyla, genera, and class levels. We then created a heat map tree of the entire hierarchy from phylum to species to examine which aspects of the tree were increased or decreased compared to the donor. Data were analyzed for significance ( P ≤ .05) by 2-tailed Student t – and Mann-Whitney U tests (GraphPad Prism 8).


Linear discriminant analysis


A stricter measure, namely linear discriminant analysis (LDA) was next used on the dataset. This analysis integrates statistical significance with biological consistency (effect size) estimation. The statistics are based on a nonparametric factorial Kruskal-Wallis rank-sum test to detect features with significant differential abundance, followed by LDA to estimate the effect size of each differentially abundant features.


Correlations between microbiome, clinical, and immune cells metrics


Correlation coefficients were used to evaluate relationships among metrics of interest.


Change in the microbiome composition over time


We examined change in OTU number over time in the phyla, classes, and genera noted to be significantly different between recipients and donors at baseline. P values were calculated between the donors and recipients at various time points using 2-tailed independent Student t -tests.


RESULTS


STUDY POPULATION


Ten subjects were enrolled in the study ( Table 1 ). The mean age of the population was 60.4 years (range: 51-66; SD ±4.2); 3 (30%) were male; 5 (50%) were white; and 5 (50%) were Hispanic. Full Sjögren criteria were met by 5 subjects (50%), and early Sjögren criteria were met by the other 5 subjects (50%) by the PSP (n = 2) or SP1 (n = 3) protein positivity. Comorbidities included hypertension (n = 4), type 2 diabetes (n = 2), and Grave disease (n = 1). Five subjects had 1 or more comorbid autoimmune diseases (RA [n = 4], UC [n = 2], or systemic lupus erythematosus [n = 2]). GI symptoms at time of enrollment included nausea (n = 4), heartburn (n = 3), abdominal pain (n = 3), constipation (n = 5), and blood in stool (n = 1). All individuals were on a stable combination of oral and topical medications, including numerous DE therapies.



TABLE 1

Population Demographics, Comorbidities, and Medications




















































































Demographics Subjects (n = 10)
Age, y, mean ± SD (range) 60.4 ± 4.2 (51-66)
Male subjects 3 (30)
White race 5 (50)
Hispanic ethnicity 4 (40)
Past smoker 2 (20)
Current smoker 1 (10)
Comorbidities
Hypertension 4 (40)
Diabetes 2 (20)
Medications
Antihypertensive 5 (50)
Lipid lowering 1 (10)
Glucose lowering 1 (10)
Analgesic 9 (90)
Antidepressant 2 (20)
Anxiolytic 2 (20)
Antihistamine 2 (20)
Oral antibiotics 1 (10)
Topical medications
Artificial tears 10 (100)
Topical cyclosporine 0.05% 6 (60)
Lifitegrast 5% 3 (30)
Autologous serum tears 3 (30)
Topical corticosteroid 2 (20)
Topical antibiotic 1 (10)
Punctal plug 4 (40)

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Jan 3, 2022 | Posted by in OPHTHALMOLOGY | Comments Off on Fecal Microbial Transplant in Individuals With Immune-Mediated Dry Eye

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