Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

BACKGROUND: Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical, host-derived regulators of the microbiota. However, mechanisms that support microbiota homeostasis in response to inflammatory stimuli, such as supraphysiologic oxygen, remain unclear. RESULTS: We show that supraphysiologic oxygen exposure to neonatal mice, or direct exposure of intestinal organoids to supraphysiologic oxygen, suppresses the intestinal expression of AMPs and alters intestinal microbiota composition. Oral supplementation of the prototypical AMP lysozyme to hyperoxia-exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. CONCLUSIONS: Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury in newborns. Together, these data support that intestinal AMPs modulate lung injury and repair. Video Abstract.

The fungal intestinal microbiota predict the development of bronchopulmonary dysplasia in very low birthweight newborns.

RATIONALE: Bronchopulmonary dysplasia (BPD) is the most common morbidity affecting very preterm infants. Gut fungal and bacterial microbial communities contribute to multiple lung diseases and may influence BPD pathogenesis. METHODS: We performed a prospective, observational cohort study comparing the multikingdom fecal microbiota of 144 preterm infants with or without moderate to severe BPD by sequencing the bacterial 16S and fungal ITS2 ribosomal RNA gene. To address the potential causative relationship between gut dysbiosis and BPD, we used fecal microbiota transplant in an antibiotic-pseudohumanized mouse model. Comparisons were made using RNA sequencing, confocal microscopy, lung morphometry, and oscillometry. RESULTS: We analyzed 102 fecal microbiome samples collected during the second week of life. Infants who later developed BPD showed an obvious fungal dysbiosis as compared to infants without BPD (NoBPD, p = 0.0398, permutational multivariate ANOVA). Instead of fungal communities dominated by Candida and Saccharomyces, the microbiota of infants who developed BPD were characterized by a greater diversity of rarer fungi in less interconnected community architectures. On successful colonization, the gut microbiota from infants with BPD augmented lung injury in the offspring of recipient animals. We identified alterations in the murine intestinal microbiome and transcriptome associated with augmented lung injury. CONCLUSIONS: The gut fungal microbiome of infants who will develop BPD is dysbiotic and may contribute to disease pathogenesis.

Clinical trial of a probiotic and herbal supplement for lung health.

Introduction: Dysbiosis of the gut microbiome may augment lung disease via the gut-lung axis. Proteobacteria may contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic inflammation. To study the effects of probiotics across the gut-lung axis, we sought to determine if a Lactobacillus probiotic and herbal blend was safe and well-tolerated in healthy volunteers and asthmatic patients. Methods: We conducted a 1-month randomized, open-label clinical trial in Cork, Ireland with healthy and asthmatic patients who took the blend twice a day. The primary endpoint was safety with exploratory endpoints including quality of life, lung function, gut microbiome ecology, and inflammatory biomarkers. Results: All subjects tolerated the blend without adverse events. Asthmatic subjects who took the blend showed significant improvements in lung function as measured by forced expiratory volume and serum short chain fatty acid levels from baseline to Week 4. The gut microbiome of asthmatic subjects differed significantly from controls, with the most prominent difference in the relative abundance of the proteobacteria Escherichia coli. Administration of the probiotic maintained overall microbial community architecture with the only significant difference being an increase in absolute abundance of the probiotic strains measured by strain-specific PCR. Conclusion: This study supports the safety and efficacy potential of a Lactobacillus probiotic plus herbal blend to act on the gut-lung axis. However, due to the lack of a control group, a longer blinded, placebo-controlled study will be warranted to confirm the efficacy improvements observed in this trial. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT05173168.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair. In Brief: Using a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota. Highlights: Supraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs).Intestinal AMP expression has an inverse relationship with the severity of lung injury.AMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury.AMPs may mediate a gut-lung axis that modulates lung injury.

COVID-19 bacteremic co-infection is a major risk factor for mortality, ICU admission, and mechanical ventilation.

BACKGROUND: Recent single-center reports have suggested that community-acquired bacteremic co-infection in the context of Coronavirus disease 2019 (COVID-19) may be an important driver of mortality; however, these reports have not been validated with a multicenter, demographically diverse, cohort study with data spanning the pandemic. METHODS: In this multicenter, retrospective cohort study, inpatient encounters were assessed for COVID-19 with community-acquired bacteremic co-infection using 48-h post-admission blood cultures and grouped by: (1) confirmed co-infection [recovery of bacterial pathogen], (2) suspected co-infection [negative culture with ≥ 2 antimicrobials administered], and (3) no evidence of co-infection [no culture]. The primary outcomes were in-hospital mortality, ICU admission, and mechanical ventilation. COVID-19 bacterial co-infection risk factors and impact on primary outcomes were determined using multivariate logistic regressions and expressed as adjusted odds ratios with 95% confidence intervals (Cohort, OR 95% CI, Wald test p value). RESULTS: The studied cohorts included 13,781 COVID-19 inpatient encounters from 2020 to 2022 in the University of Alabama at Birmingham (UAB, n = 4075) and Ochsner Louisiana State University Health-Shreveport (OLHS, n = 9706) cohorts with confirmed (2.5%), suspected (46%), or no community-acquired bacterial co-infection (51.5%) and a comparison cohort consisting of 99,170 inpatient encounters from 2010 to 2019 (UAB pre-COVID-19 pandemic cohort). Significantly increased likelihood of COVID-19 bacterial co-infection was observed in patients with elevated ≥ 15 neutrophil-to-lymphocyte ratio (UAB: 1.95 [1.21-3.07]; OLHS: 3.65 [2.66-5.05], p < 0.001 for both) within 48-h of hospital admission. Bacterial co-infection was found to confer the greatest increased risk for in-hospital mortality (UAB: 3.07 [2.42-5.46]; OLHS: 4.05 [2.29-6.97], p < 0.001 for both), ICU admission (UAB: 4.47 [2.87-7.09], OLHS: 2.65 [2.00-3.48], p < 0.001 for both), and mechanical ventilation (UAB: 3.84 [2.21-6.12]; OLHS: 2.75 [1.87-3.92], p < 0.001 for both) across both cohorts, as compared to other risk factors for severe disease. Observed mortality in COVID-19 bacterial co-infection (24%) dramatically exceeds the mortality rate associated with community-acquired bacteremia in pre-COVID-19 pandemic inpatients (5.9%) and was consistent across alpha, delta, and omicron SARS-CoV-2 variants. CONCLUSIONS: Elevated neutrophil-to-lymphocyte ratio is a prognostic indicator of COVID-19 bacterial co-infection within 48-h of admission. Community-acquired bacterial co-infection, as defined by blood culture-positive results, confers greater increased risk of in-hospital mortality, ICU admission, and mechanical ventilation than previously described risk factors (advanced age, select comorbidities, male sex) for COVID-19 mortality, and is independent of SARS-CoV-2 variant.

Microbial-induced Redox Imbalance in the Neonatal Lung Is Ameliorated by Live Biotherapeutics.

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants. Hyperoxia exposure and microbial dysbiosis are contributors to BPD development. However, the mechanisms linking pulmonary microbial dysbiosis to worsening lung injury are unknown. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates oxidative stress responses and modulates hyperoxia-induced lung injury. We hypothesized that airway dysbiosis would attenuate Nrf2-dependent antioxidant function, resulting in a more severe phenotype of BPD. Here, we show that preterm infants with a Gammaproteobacteria-predominant dysbiosis have increased endotoxin in tracheal aspirates, and mice monocolonized with the representative Gammaproteobacteria Escherichia coli show increased tissue damage compared with germ-free (GF) control mice. Furthermore, we show Nrf2-deficient mice have worse lung structure and function after exposure to hyperoxia when the airway microbiome is augmented with E. coli. To confirm the disease-initiating potential of airway dysbiosis, we developed a novel humanized mouse model by colonizing GF mice with tracheal aspirates from human infants with or without severe BPD, producing gnotobiotic mice with BPD-associated and non-BPD-associated lung microbiomes. After hyperoxia exposure, BPD-associated mice demonstrated a more severe BPD phenotype and increased expression of Nrf2-regulated genes, compared with GF and non-BPD-associated mice. Furthermore, augmenting Nrf2-mediated antioxidant activity by supporting colonization with Lactobacillus species improved dysbiotic-augmented lung injury. Our results demonstrate that a lack of protective pulmonary microbiome signature attenuates an Nrf2-mediated antioxidant response, which is augmented by a respiratory probiotic blend. We anticipate antioxidant pathways will be major targets of future microbiome-based therapeutics for respiratory disease.

Efficacy of a Probiotic and Herbal Supplement in Models of Lung Inflammation.

BACKGROUND: Gut microbiome dysbiosis is associated with lung disease through the gut-lung axis. Abundant proteobacteria increase MMP-9 and contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue injury, and perpetuation of chronic lung disease. We sought to determine if a scientifically formulated probiotic and herbal supplement could attenuate neutrophilic inflammation and improve lung structure and function in models of lung inflammation. METHODS: For in vitro experiments, epithelial cells exposed to proteobacteria were treated with resB-a blend of three probiotic Lactobacillus strains and turmeric, holy basil, and vasaka herbal extracts. For in vivo experimentation, mice exposed to pulmonary proteobacteria-derived lipopolysaccharide were treated by gavage with resB. RESULTS: In vitro, the bacterial and herbal components of resB decreased activity of the MMP-9 pathway. Mice exposed to LPS and pre- and post-treated with resB had decreased neutrophil recruitment and inflammatory biomarkers in bronchoalveolar lavage fluid, serum, and lung tissue compared to untreated mice. CONCLUSIONS: This study describes the mechanisms and efficacy of probiotic and herbal blend in pre-clinical models of lung injury and inflammation.

Hospital and Neurodevelopmental Outcomes in Nano-Preterm Infants Receiving Invasive vs Noninvasive Ventilation at Birth.

Importance: Infants with gestational age between 22 0/7 and 23 6/7 weeks (referred to as nano-preterm infants) are at very high risk of adverse outcomes. Noninvasive respiratory support at birth improves outcomes in infants born at 24 0/7 to 27 6/7 weeks' gestational age. Evidence is limited on whether similar benefits of non-invasive respiratory support at birth extend to nano-preterm infants. Objective: To evaluate the hypothesis that intubation at 10 minutes or earlier after birth is associated with a higher incidence of bronchopulmonary dysplasia (BPD) or death by 36 weeks' postmenstrual age (PMA) in nano-preterm infants. Design, Setting, and Participants: This observational cohort study included all nano-preterm infants at a level IV neonatal intensive care unit who were delivered from January 1, 2014, to June 30, 2021. Infants receiving palliative or comfort care at birth were excluded. Exposures: Infants were grouped based on first intubation attempt timing after birth (>10 minutes after birth and ≤10 minutes as noninvasive and invasive respiratory support at birth groups, respectively). Main Outcomes and Measures: The primary outcome was the composite outcome of BPD (physiological definition) or death by 36 weeks' PMA. Results: All 230 consecutively born, eligible nano-preterm infants were included, of whom 88 (median [IQR] gestational age, 23.6 [23.4-23.7] weeks; 45 [51.1%] female; 54 [62.1%] Black) were in the noninvasive respiratory support at birth group and 142 (median [IQR] gestational age, 23.0 [22.4-23.3] weeks; 71 [50.0%] female; 94 [66.2%] Black) were in the invasive respiratory support at birth group. The incidence of BPD or death by 36 weeks' PMA did not differ between the noninvasive and invasive respiratory support groups (83 of 88 [94.3%] in the noninvasive group vs 129 of 142 [90.9%] in the invasive group; adjusted odds ratio, 2.09; 95% CI, 0.60-7.25; P = .24). Severe intraventricular hemorrhage or death by 36 weeks' PMA was lower in the invasive respiratory support at birth group (adjusted odds ratio, 2.20; 95% CI, 1.07-4.51; P = .03). Conclusions and Relevance: This cohort study's findings suggest that noninvasive respiratory support in the first 10 minutes after birth is feasible but is not associated with a decrease in the risk of BPD or death compared with intubation and early surfactant delivery in nano-preterm infants.

Duration of noninvasive respiratory support and risk for bronchopulmonary dysplasia or death.

OBJECTIVE: To determine whether the duration of noninvasive respiratory support exposure is associated with bronchopulmonary dysplasia (BPD) or death in preterm infants. METHODS: Multicenter, retrospective study of infants born at <29 weeks' gestation. The association between days on noninvasive respiratory support and BPD or death was determined using instrumental variable techniques and generalized propensity score matching to account for potential confounding by illness severity. RESULTS: Among 6268 infants 36% developed BPD or died. The median duration of noninvasive respiratory support was 18 days. There was inconsistency in the association between noninvasive support and BPD or death when analyzed by instrumental variable techniques (Average Marginal Effect -0.37; 95% CI -1.23 to 0.50) and generalized propensity score matching (Average Marginal Effect 0.46; 95% CI 0.33 to 0.60). CONCLUSION: Findings on the association between duration of exposure to noninvasive respiratory support and the development of BPD or death were inconclusive. GOV ID: Generic Database:NCT00063063.

A Quality Improvement Bundle to Improve Outcomes in Extremely Preterm Infants in the First Week.

OBJECTIVES: Our objective with this quality improvement initiative was to reduce rates of severe intracranial hemorrhage (ICH) or death in the first week after birth among extremely preterm infants. METHODS: The quality improvement initiative was conducted from April 2014 to September 2020 at the University of Alabama at Birmingham's NICU. All actively treated inborn extremely preterm infants without congenital anomalies from 22 + 0/7 to 27 + 6/7 weeks' gestation with a birth weight ≥400 g were included. The primary outcome was severe ICH or death in the first 7 days after birth. Balancing measures included rates of acute kidney injury and spontaneous intestinal perforation. Outcome and process measure data were analyzed by using p-charts. RESULTS: We studied 820 infants with a mean gestational age of 25 + 3/7 weeks and median birth weight of 744 g. The rate of severe ICH or death in the first week after birth decreased from the baseline rate of 27.4% to 15.0%. The rate of severe ICH decreased from a baseline rate of 16.4% to 10.0%. Special cause variation in the rate of severe ICH or death in the first week after birth was observed corresponding with improvement in carbon dioxide and pH targeting, compliance with delayed cord clamping, and expanded use of indomethacin prophylaxis. CONCLUSIONS: Implementation of a bundle of evidence-based potentially better practices by using specific electronic order sets was associated with a lower rate of severe ICH or death in the first week among extremely preterm infants.

The gut microbiome of extremely preterm infants randomized to the early progression of enteral feeding.

BACKGROUND: Early progression of feeding could influence the development of the gut microbiome. METHODS: We collected fecal samples from extremely preterm infants randomized to receive either early (feeding day 2) or delayed (feeding day 5) feeding progression. After study completion, we compared samples obtained at three different time points (week 1, week 2, and week 3) to determine longitudinal differences in specific taxa between the study groups using unadjusted and adjusted negative binomial and zero-inflated mixed models. Analyses were adjusted for a mode of delivery, breastmilk intake, and exposure to antibiotics. RESULTS: We analyzed 137 fecal samples from 51 infants. In unadjusted and adjusted analyses, we did not observe an early transition to higher microbial diversity within samples (i.e., alpha diversity) or significant differences in microbial diversity between samples (i.e., beta diversity) in the early feeding group. Our longitudinal, single-taxon analysis found consistent differences in the genera Lactococcus, Veillonella, and Bilophila between groups. CONCLUSIONS: Differences in single-taxon analyses independent of the mode of delivery, exposure to antibiotics, and breastmilk feeding suggest potential benefits of early progression of enteral feeding volumes. However, this dietary intervention does not appear to increase the diversity of the gut microbiome in the first 28 days after birth. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02915549. IMPACT: Early progression of enteral feeding volumes with human milk reduces the duration of parenteral nutrition and the need for central venous access among extremely preterm infants. Early progression of enteral feeding leads to single-taxon differences in longitudinal analyses of the gut microbiome, but it does not appear to increase the diversity of the gut microbiome in the first 28 days after birth. Randomization in enteral feeding trials creates appealing opportunities to evaluate the effects of human milk diets on the gut microbiome.

Prolyl endopeptidase contributes to early neutrophilic inflammation in acute myocardial transplant rejection.

Altered inflammation and tissue remodeling are cardinal features of cardiovascular disease and cardiac transplant rejection. Neutrophils have increasingly been understood to play a critical role in acute rejection and early allograft failure; however, discrete mechanisms that drive this damage remain poorly understood. Herein, we demonstrate that early acute cardiac rejection increases allograft prolyl endopeptidase (PE) in association with de novo production of the neutrophil proinflammatory matrikine proline-glycine-proline (PGP). In a heterotopic murine heart transplant model, PGP production and PE activity were associated with early neutrophil allograft invasion and allograft failure. Pharmacologic inhibition of PE with Z-Pro-prolinal reduced PGP, attenuated early neutrophil graft invasion, and reduced proinflammatory cytokine expression. Importantly, these changes helped preserve allograft rejection-free survival and function. Notably, within 2 independent patient cohorts, both PGP and PE activity were increased among patients with biopsy-proven rejection. The observed induction of PE and matrikine generation provide a link between neutrophilic inflammation and cardiovascular injury, represent a potential target to reduce allogenic immune responses, and uncover a mechanism of cardiovascular disease that has been previously unrecognized to our knowledge.

MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha.

BACKGROUND: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD). METHODS: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants. RESULTS: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation. CONCLUSIONS: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Neutrophilic inflammation during lung development disrupts elastin assembly and predisposes adult mice to COPD.

Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in the airway epithelium, we found that a brief period of inflammation during the saccular stage (P3-P5) but not alveolar stage (P10-P12) of lung development disrupted elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progressed through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation downregulated elastin and fibulin-5 expression by saccular-stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. Although neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early-life predisposition to COPD.

Bronchopulmonary dysplasia is associated with reduced oral nitrate reductase activity in extremely preterm infants.

Oral microbiome mediated nitrate reductase (NR) activity regulates nitric oxide (NO) bioavailability and signaling. While deficits in NO-bioavailability impact several morbidities of extreme prematurity including bronchopulmonary dysplasia (BPD), whether oral NR activity is associated with morbidities of prematurity is not known. We characterized NR activity in extremely preterm infants from birth until 34 weeks' post menstrual age (PMA), determined whether changes in the oral microbiome contribute to changes in NR activity, and determined whether changes in NR activity correlated with disease. In this single center prospective cohort study (n = 28), we observed two surprising findings: (1) NR activity unexpectedly peaked at 29 weeks' PMA (p < 0.05) and (2) when infants were stratified for BPD status, infants who developed BPD had significantly less NR activity at 29 weeks' PMA compared to infants who did not develop BPD. Oral microbiota and NR activity may play a role in BPD development in extremely preterm infants, indicating potential for disease prediction and therapeutic targeting.

Airway nitrite is increased in extremely preterm infants with bronchopulmonary dysplasia.

RATIONALE: Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity and significantly contributes to mortality and morbidity with few predictive biomarkers. Given that nitrites have been implicated in pathways associated with lung disease, we hypothesized that nitrite levels would be altered in the airways of premature infants diagnosed with BPD. METHODS: This was a prospective cohort study of extremely low birth infants (< 28 weeks' gestation) at the University of Alabama at Birmingham. Nitrite levels from tracheal aspirates (TAs) were compared between intubated and ventilated infants with BPD and gestation matched full term (FT) controls. TA derived nitrite levels from day one after birth were also compared between preterm infants who did and did not develop BPD. RESULTS: Infants with BPD were found to have significantly elevated nitrite levels in their tracheal aspirates compared to gestation matched FT controls (p < 0.05). There was a trend for increased nitrite levels on postnatal day one in infants that developed BPD compared to infants that did not develop BPD (p = 0.05). CONCLUSIONS: In conclusion, nitrite levels are significantly increased in airways of infants with BPD. Data from a larger cohort are needed to further support the utility of nitrite for BPD prediction. TRIAL REGISTRATION: Not applicable.

The closed eye harbours a unique microbiome in dry eye disease.

Dry eye affects millions of individuals. In experimental models, dry eye disease is associated with T helper cell 17-mediated inflammation of the ocular surface that may cause persistent damage to the corneal epithelium. However, the initiating and perpetuating factors associated with chronic inflammation of the ocular surface remain unclear. The ocular microbiota alters ocular surface inflammation and may influence dry eye disease development and progression. Here, we collected serial samples of tears on awakening from sleep, closed eye tears, during a randomized clinical trial of a non-pharmaceutical dry eye therapy and used 16S rRNA metabarcoding to characterize the microbiome. We show the closed dry eye microbiome is distinct from the healthy closed eye microbiome, and that the microbiome remains distinct despite daily saline eye wash upon awakening. The ocular microbiome was described only recently, and this report implicates a distinct microbiome in ocular disease development. Our findings suggest an interplay between microbial commensals and inflammation on the ocular surface. This information may inform future studies of the pathophysiological mechanisms of dry eye disease.