Role of Microbiota in Strengthening Ocular Mucosal Barrier Function Through Secretory IgA

Abirami Kugadas, Quentin Wright, Jennifer Geddes-McAlister, Mihaela Gadjeva, Abirami Kugadas, Quentin Wright, Jennifer Geddes-McAlister, Mihaela Gadjeva

Abstract

Purpose: The purpose of this study was to evaluate mechanisms controlling secretory IgA (SIgA) production, thereby ensuring maintenance of ocular surface health.

Methods: To determine whether the presence of specific gut commensal species regulates SIgA levels and IgA transcripts in the eye-associated lymphoid tissues (EALT), specific-pathogen-free (SPF) Swiss Webster (SW) mice were treated with antibiotic cocktails, germ-free (GF) SW mice were reconstituted with diverse commensal gut microbiota, or monocolonized with gut-specific commensals. Proteomic profiling and quantitative real-time polymerase chain reaction (qRT-PCR) were used to quantify SIgA and IgA levels. 16S rDNA sequencing was carried out to characterize commensal microbiota.

Results: Commensal presence regulated ocular surface SIgA levels and mRNA IgA transcripts in EALT. Oral antibiotic cocktail intake significantly reduced gut commensal presence, while maintaining ocular surface commensal levels reduced SIgA and IgA transcripts in EALT. Analysis of gut microbial communities revealed that SPF SW mice carried abundant Bacteroides organisms when compared to SPF C57BL6/N mice, with B. acidifaciens being the most prominent species in SPF SW mice. Monocolonization of GF SW mice with B. acidifaciens, a strict gut anaerobe, resulted in significant increase of IgA transcripts in the EALT, implying generation of B-cell memory.

Conclusions: These data illustrated a "gut-eye" axis of immune regulation. Exposure of the host to gut commensal species may serve as a priming signal to generate B-cell repertoires at sites different from the gut, such as EALT, thereby ensuring broad protection.

Figures

Figure 1
Figure 1
Quantitative LC-MS/MS analysis reveals distinct ocular surface proteomic signatures in SPF SW and SPF C57BL/6N mice. (A) Functional analysis of ocular surface proteomes in SPF C57BL/6N and SPF SW mice, based on relative representation of biological processes. (B) Volcano plot differentially expressed ocular surface proteins in SPF SW and SPF C57BL/6N mice. Proteins with significant change in relative abundances in SPF SW mice are shown in red, while those in the SPF C57BL6/N mice are depicted in blue (see Supplementary Table S1 for protein ID).
Figure 2
Figure 2
IgA transcript levels differ in genetically distinct strains of mice. CLNs (A) and LGs (B) were harvested from 8- to 10-week-old SPF SW and SPF C57BL/6N mice, processed for total RNA, and analyzed for IgA gene expression with qPCR. Expression was determined as n-fold change induction as compared with the GAPDH housekeeping gene. The significance of differences, based on a Student's t-test, relative to an SPF C57BL/6N control was plotted. Data are representative of two separate experiments including four to seven mice per group. The data demonstrate elevated levels of IgA transcripts in SPF SW EALT.
Figure 3
Figure 3
Gut commensal bacteria influence LG IgA transcription. (A) LG tissue was recovered from SPF SW (n = 7) and ABX-treated SPF SW (n = 7) mice and analyzed for IgA gene expression. Data were plotted as median values with range, and P values were determined by the Mann-Whitney test. (B) GF SW mice were reconstituted with SPF C57BL/6N or SPF SW gut homogenate. LGs were extracted and analyzed for IgA gene expression. Expression was determined as n-fold change induction as compared with the GAPDH housekeeping gene. Data were analyzed by 1-way ANOVA followed by Dunnett's comparisons. Five mice per group were analyzed. Data show that the GF SW mice reconstituted with SPF SW gut commensals induce higher levels of LG IgA transcripts than GF SW mice reconstituted with SPF C57BL/6N gut commensals.
Figure 4
Figure 4
SPF SW mice show richer commensal gut composition than SPF C57BL/6N mice. (A) 16S sequencing analysis of gut commensal communities of SPF SW (n = 3) and SPF C57BL/6N (n = 3) mice show significant differences in the abundance of specific commensal genera by Shannon index of diversity. (B) Plotted data represent 95% of recovered 16S rDNA sequences per genotype. Data are presented as mean percentage frequencies for each genus. P values were generated by using Student's t-test and the Bonferroni correction for multiple comparisons was applied, P

Figure 5

IgA increases at multiple mucosal…

Figure 5

IgA increases at multiple mucosal sites after recolonization of GF SW mice with…

Figure 5
IgA increases at multiple mucosal sites after recolonization of GF SW mice with Bacteroides acidifaciens. GF SW mice (n = 5–7) were orally gavaged with (1 × 108 CFU/mL) B. acidifaciens and kept in GF housing for 21 days. (A) Significance of changes in the colon IgA gene transcripts over time was determined by 1-way ANOVA followed by Dunn's comparison test. (B) Stool samples were assayed for SIgA by ELISA. Significance of changes in gut SIgA over time was determined by 1-way ANOVA followed by Dunnett's comparison test. (C) Changes in IgA transcript levels in the small-intestine samples. Significance was determined by Student's t-test. (D) Significance of changes in LG IgA transcripts over time was determined by 1-way ANOVA followed by Dunnett's comparison test. (E) Pooled eyewash samples were collected from mice and analyzed for IgA by ELISA. Significance of changes in eyewash SIgA over time was determined by 1-way ANOVA followed by Dunn's comparison test. Cumulatively, the data show that gut reconstitution of GF SW mice with B. acidifaciens induced a robust gut and ocular IgA transcription.

Figure 6

Systemic IL-1β blockade modulates IgA…

Figure 6

Systemic IL-1β blockade modulates IgA production in the LG. (A) ELISA for serum…

Figure 6
Systemic IL-1β blockade modulates IgA production in the LG. (A) ELISA for serum IL-1β levels in GF SW and SPF SW mice. Significance was determined by a Student's t-test. Cervical lymph nodes (B) and LGs (C) were harvested from SPF SW mice after systemic IL-1β antibody treatment and analyzed for IgA gene expression by using qRT-PCR. Significance was determined by a Student's t-test. Control mice received isotype control treatment. (D) Pooled eyewash samples were collected 24 hours after administration of an IL-1β blockade and were assayed for SIgA by ELISA. Significance was determined by a Student's t-test. Cumulatively, data show that ocular SIgA concentration at steady state depends on IL-1β signaling.
Figure 5
Figure 5
IgA increases at multiple mucosal sites after recolonization of GF SW mice with Bacteroides acidifaciens. GF SW mice (n = 5–7) were orally gavaged with (1 × 108 CFU/mL) B. acidifaciens and kept in GF housing for 21 days. (A) Significance of changes in the colon IgA gene transcripts over time was determined by 1-way ANOVA followed by Dunn's comparison test. (B) Stool samples were assayed for SIgA by ELISA. Significance of changes in gut SIgA over time was determined by 1-way ANOVA followed by Dunnett's comparison test. (C) Changes in IgA transcript levels in the small-intestine samples. Significance was determined by Student's t-test. (D) Significance of changes in LG IgA transcripts over time was determined by 1-way ANOVA followed by Dunnett's comparison test. (E) Pooled eyewash samples were collected from mice and analyzed for IgA by ELISA. Significance of changes in eyewash SIgA over time was determined by 1-way ANOVA followed by Dunn's comparison test. Cumulatively, the data show that gut reconstitution of GF SW mice with B. acidifaciens induced a robust gut and ocular IgA transcription.
Figure 6
Figure 6
Systemic IL-1β blockade modulates IgA production in the LG. (A) ELISA for serum IL-1β levels in GF SW and SPF SW mice. Significance was determined by a Student's t-test. Cervical lymph nodes (B) and LGs (C) were harvested from SPF SW mice after systemic IL-1β antibody treatment and analyzed for IgA gene expression by using qRT-PCR. Significance was determined by a Student's t-test. Control mice received isotype control treatment. (D) Pooled eyewash samples were collected 24 hours after administration of an IL-1β blockade and were assayed for SIgA by ELISA. Significance was determined by a Student's t-test. Cumulatively, data show that ocular SIgA concentration at steady state depends on IL-1β signaling.

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