Fig 1.
GF mice have decreased levels of innate and adaptive immune effectors at the ocular surface.
A. Significantly decreased levels of complement proteins at the ocular surface in GF mice compared to SPF mice. B. Significantly decreased levels of iron binding protein serotransferrin at the ocular surface. C. Decreased levels of immunoglobulins at the ocular surface. D. Decreased levels of neutrophil-derived peptides at the ocular surface. Significant differences were detected in the levels of nuclear cytosolic protein 1 (Ncf1) and 2 (Ncf 2), myeloid bactenecin (ngp), neutrophil galatinase associated lipocalin (Lcn2), and chitinase-3-like protein 1. Ocular surface washes were pooled from GF (n = 5) and SPF mice (n = 5). Two-three biological replicates were analyzed per experiment. Five μg total protein were digested with trypsin, peptides were labeled, multiplexed with TMT, and quantified using LC-MS3. p-values were calculated using Benjamini-Hochberg FDR correction.
Fig 2.
GF mice are more susceptible to ocular P. aeruginosa–induced keratitis compared to SPF mice.
A. P. aeruginosa 6294 CFU/cornea (left) and pathology scores (right) 24 h following infection. Groups of GF (n = 11) and SPF SW (n = 14) mice were infected with 1×107 CFU P. aeruginosa per eye. Data were pooled from two independent experiments performed under comparable conditions. p-values were generated using Student’s t-test. B. Hematoxylin-eosin staining of sections derived from GF and SPF SW mice. Images are taken at 10x magnification to illustrate pathological alterations during infection. Data are representative images taken from the infected eyes of GF mice (n = 4) and SPF (n = 4) mice. The quantification of histopathological changes confirmed increased pathology in GF corneas when compared to SPF. p-values by Mann-Whitney test.
Fig 3.
Inflammatory responses in the corneas of GF SW mice are increased when compared to SPF mice infected with P. aeruginosa strain 6294.
Groups of 14 GF mice and 14 SPF mice were infected with 1×107 CFU P. aeruginosa placed onto scratch-injured eyes. Corneas were harvested at 24 h after infection, washed in F12 media, homogenized in PBS containing a mix of protease inhibitors and supplemented with 0.5% Triton to disrupt plasma membranes. The levels of cytokines in corneal lysates were measured using a Meso Scale Discovery (MSD) multiplex assay and compared with Bonferroni’s correction for multiple comparisons (p<0.008). IL-12p40, IL-6 and KC were significantly increased in GF mice when compared to SPF mice p = 0.0001, p = 0.0001, and p = 0.0001, respectively. Data pooled from two experiments performed under comparable conditions.
Fig 4.
Neutrophil recruitment to the corneas of infected GF SW mice is increased at the peak of infection.
Ocular surface washes were collected from P. aeruginosa-infected GF (n = 5) and SPF SW (n = 5) mice. 5 μg total protein was in-gel digested, trypsinized, labeled, and profiled using LC-MS/MS. Significantly upregulated proteins in the infected GF mice were visualized using String-based web software and revealed enrichment for proteins associated with phagocytosis (FDR 0.000525), phagosome (FDR 0.003), leukocyte trafficking (FDR 0.02) and high degree of protein-protein interaction (PPI enrichment p value 7.55e-15).
Fig 5.
Topical gentamycin treatment reduces the resistance to P. aeruginosa-induced keratitis.
A. Commensal conjunctival presence in SPF mice (n = 10) is significantly higher than in mice (n = 10) treated topically with gentamycin ointment for 3 days prior to the infectious challenge. Data were pooled from two independent experiments. Groups were compared with one-way ANOVA, p = 0.01. B. Detection of bacterial growth from conjunctival swabs on a blood agar plate. The left side of the plate was seeded with 10 μl/spot suspension derived from the conjunctival swabs of control mice, the right side of the plate was seeded with 10 μl/spot suspension derived from the conjunctival swabs of Gentamycin-treated mice. C. Corneal bacterial burden after challenge with P. aeruginosa PAO1 is moderately but significantly increased in the Gentamycin–treated cohort. Data were pooled from three performed experiments. Unpaired Student’s t-test. D. Pathology scores. p-values are derived by Mann-Whitney test.
Fig 6.
Oral antibiotic treatment significantly reduces the resistance to P. aeruginosa-induced infection.
A. Aerobic and anaerobic gut commensal bacterial burden in SPF mice after oral antibiotic treatment shows significant decrease in gut commensal presence. Circles indicate individual animals. p-values by unpaired Student’s t-test. B. Commensal conjunctival presence is not affected in mice treated with oral antibiotics. Circles indicate individual animals. C. Pathology scores and corneal bacterial burden at 24h after infection. Groups of 10 SPF SW mice and 10 ABX mice were infected with 1×107 CFU P. aeruginosa PAO1. Mouse corneas were harvested at 24 h after infection. ABX mice had significantly higher pathology scores and bacterial burdens indicating reduced resistance to infection. p- values by Student’s t-test (CFU) and Mann-Whitney test (pathology). Data were pooled from two experiments performed. D. Corneal bacterial P. aeruginosa 6294 burden measured at 24h after the infectious challenge in cohorts of SPF mice, mice treated locally with gentamycin and orally with antibiotics (gent/ABX), and mice treated orally with antibiotics. ABX mice treated with gentamycin showed comparable bacterial levels to the ABX mice that received no additional topical antibiotic treatment. Data were pooled from two experiments performed. The circles represent individual mice. p-values by one-way ANOVA (p<0.0001) followed by Turkey’s multiple comparisons test.
Fig 7.
PMNs derived from either GF mice or mice treated with oral antibiotics have significantly decreased bactericidal activities against P. aeruginosa and altered transcriptomic signatures.
A. Percent P. aeruginosa PAO1 killing by GF PMNs and SPF SW-derived PMNs in vitro (p = 0.01, Unpaired Student t-test). Bone marrow-derived mature GF and SPF SW PMNs were exposed to P. aeruginosa PAO1 for 90 min at 37°C. Upon completion of incubation, an aliquot from the reaction mixture was plated to quantify the remaining PAO1. Neutrophils derived from animals without commensal microbiota display significantly reduced bactericidal capacity against P. aeruginosa. B. Percent of P. aeruginosa PAO1 killing by SPF SW-derived PMNs and PMNs purified from mice treated with oral antibiotic cocktail in vitro (p = 0.002, unpaired Student t-test). Neutrophils derived from mice that received oral antibiotics display significantly reduced bactericidal capacity against P. aeruginosa, illustrating that microbiota-derived signals promote neutrophil function. C. GF-derived neutrophils have significantly different transcriptional signature when compared to SPF-derived neutrophils. Shown are the transcriptomic profiles of differentially expressed genes in GF- and SPF-derived PMNs obtained by RNAseq analysis and the predicted pathways involved in PMN maturation in a heat map of the differentially expressed genes in GF- and SPF-derived PMNs. Hierarchical cluster analysis was performed on CLC Genomics workbench on subset of transcripts that show differential presence between the GF- and SPF-derived neutrophils (≥ 1.5-fold; p<0.05). The same subset was used to predict the potential upstream regulators by IPA to identify pathways that are involved in priming neutrophils for optimal function.
Fig 8.
Commensal presence promotes IL-1ß release during infection.
A. Differential presence of IL-1ß transcripts in the conjunctival tissues of GF and SPF SW mice. Quantitative RT-PCR for IL-1ß and IL-1α transcripts were carried out using conjunctival tissues from GF and SPF mice. GF conjunctival tissues have significantly lower levels of IL-1ß transcripts when compared to SPF-derived tissues. Data were pooled from two independent experiments and plotted as median values with range. The circles represent individual mice. p-values by Student’s t-test. Bonferroni correction for multiple comparisons p = 0.025.B. IL-1β protein levels in the corneal tissues of infected GF and SPF mice with P. aeruginosa 6294. Groups of GF (n = 5) and SPF-SW (n = 5) mice were infected with 1×107 CFU P. aeruginosa 6294 onto eyes. Infected GF mice mount significantly less IL-1ß when compared to SPF mice. Data were pooled from two experiments. p-values by Student’s t-test. C. IL-1ß levels measured by ELISA in the corneal tissues of either mice treated topically with gentamycin or control mice infected with P. aeruginosa PA01. Topical gentamycin treatment significantly reduced the levels of IL-1ß released during infection. Data were median values with range. p-values by Student’s t-test. D. Neutralization of IL-1ß increased corneal bacterial burden in the resistant SPF SW mice (n = 6) when compared to the control mice (n = 6). p-values by Student’s t-test. Data are pooled from two performed experiments.
Fig 9.
CNS sp restore resistance to ocular P. aerugiunosa 6294–induced keratitis.
A. P. aeruginosa 6294 CFU/cornea (left) and pathology scores (right) 24 h following infection. Groups of GF (n = 8), SPF-SW (n = 10), HMB (n = 10), and MMB (n = 10) mice were infected with 107 CFU per eye. Data were pooled from two performed experiments. The bacterial burden data and pathology scores were analyzed with one-way ANOVA (p<0.0001) followed by Kruskal-Wallis multiple comparisons test. The individual circles represent individual animals. Data suggest that reconstituting GF mice with either human-derived or mouse-derived gut commensals restores resistance to infection. B. Monocolonizing GF mice with CNS sp. by topical application of CNS sp. suspension onto the eye restores resistance to infection. Quantification of the levels of CNS sp recovered from the conjunctival swabs one week and 12 days post-reconstitution. Quantification of the CNS sp. in the fecal pellets, demonstrating stable monoassociation. C. Neutrophils derived from monocolonized GF mice show comparable bactericidal activity against P. aeruginosa PAO1 as SPF-derived PMNs. p-values by one-way ANOVA (p<0.0001) followed by Kruskal-Wallis multiple comparisons test. The individual sample values are plotted as circles. D. Conjunctival CNS sp. presence promotes IL-1ß transcription. Conjunctival tissues from GF (n = 5) and monocolonized GF mice with CNS sp. (n = 3) were collected, total RNA purified, and the levels of IL-1ß transcripts quantified with RT-PCR. The levels of transcripts were compared using one-way ANOVA followed by Turkey’s multiple comparisons test. Significant p<0.05 values are indicated by asterisks. The tissues harvested from the monocolonized mice showed significantly increased presence of IL-1ß transcripts 12 days post-colonization. E. P. aeruginosa 6294 CFU/cornea (left) and pathology scores (right) 24 h following infection in monocolonized GF with CNS sp. (n = 6), SPF SW (n = 5), or GF (n = 10) mice. Monocolonizing of GF mice with CNS sp. restores the resistance to infection. The values for the individual animals are represented as circles. Significant differences are denoted with asterisks. p-values by one-way ANOVA followed (p = 0.0007) by ordinary one-way ANOVA multiple comparisons test. Pathology scores were compared using one-way ANOVA (p<0.0001) followed by Kruskal-Wallis multiple comparison test.