Contact lens wear in humans and mice is consistently associated with asymptomatic corneal parainflammation. Here, we tested the hypothesis that the corneal response to lens wear alone can function to protect it against bacterial adhesion enabled by superficial injury. One eye only of mT/mG-LysMcre mice (cell membranes red; Lyz2+ cells green) wore a contact lens for 4-6 days. After lens removal, mice were anesthetized and both corneas superficially-injured before bacterial inoculation with either a mouse eyelid commensal (Macrococcus epidermidis) or a corneal pathogen (Pseudomonas aeruginosa). Inoculation was repeated hourly for 4 hours under anesthesia before euthanasia. Enucleated eyes were fixed overnight, and adherent bacteria visualized using a universal 16S rRNA-targeted FISH probe (M. epidermidis) or Blue Fluorescent Protein (P. aeruginosa). Confocal imaging and Imaris software were used to quantify bacterial adhesion and location in the epithelium, and also the number, location and morphology of Lyz2+ cells. For both commensal and P. aeruginosa, prior lens wear resulted in reduced adhesion to the superficially-injured corneas (~46% and ~57% respectively). In both instances this correlated with increased numbers of corneal Lyz2+ cells. Other details differed for the two bacterial types. For the commensal, prior lens wear resulted in bacteria penetrating deeper into the epithelium versus contralateral eyes, with Lyz2+ cells extending their processes further into the epithelium and localizing closer to the cornea surface. For P. aeruginosa, prior lens wear resulted in adherent bacteria closer to the cornea surface, and Lyz2+ cells moving further away from it. Moreover, while overall Lyz2+ cell sphericity increased for P. aeruginosa with prior lens wear versus contralateral eyes, it showed no overall change for the commensal. Lyz2+ cell volume in the central cornea was decreased for P. aeruginosa with prior lens wear but increased for the commensal. Thus, corneal responses to prior lens wear can quantitively reduce bacterial adhesion to superficially-injured corneas correlating with a Lyz2+ cell response for both a commensal and a pathogen, with differences in details for the two bacterial types. How continued lens wear supersedes this protective response to promote P. aeruginosa infection pathogenesis remains to be determined, as does the relationship to the Lyz2+ cell response.

AbstractMultilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistancein situin the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium byPseudomonas aeruginosarequires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within thein situmultilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a ΔpscCmutant unable to assemble the T3SS needle traversed as efficiently as wild-typeP. aeruginosa, while a ΔexsDmutant ‘constitutively on’ for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes (ΔpscU-Lmutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducibleexsA, but not if the effector genes were also deleted (ΔpscU-LΔexoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-inducedexsA-complemented ΔpscU-Lmutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal byP. aeruginosacan involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding ofP. aeruginosapathogenesis and has relevance to development of therapeutics targeting the T3SS system.ImportanceWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations ofin vivo/in situmodels and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used byP. aeruginosato traverse an epithelial multilayerin situwere studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteriain situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

Purpose:Previously, we showed that transient receptor potential ion channels TRPA1 and TRPV1 selectively protect the cornea against bacterial adhesion, with TRPA1 countering the Gram-negative pathogen Pseudomonas aeruginosa (P. aeruginosa) and TRPV1 countering environmental bacteria. Here, we explored parameters of this specificity using a Gram-positive pathogen Staphylococcus aureus (S. aureus). Methods:Healthy corneas of C57BL/6J wild-type (WT), TRPA1 (-/-), or TRPV1 (-/-) mice were challenged with S. aureus for 4 or 6 hours. Some experiments instead/also used resiniferatoxin (RTX) to deplete TRPV1-expressing nerves, JNJ-17203212 to selectively antagonize TRPV1, or the anesthetic bupivacaine to inhibit nerve firing. Adherent bacteria were quantified using fluorescence in situ hybridization (FISH) labeling (16S rRNA-targeted probe). Lyz2+, CD11c+, and CD45+ cells were visualized/quantified using hybrid mT/mG + LysMcre mice (red cell membranes; Lyz2+-GFP), CD11c+-YFP mice, and anti-CD45-antibody, respectively. Results:Corneas of TRPV1 (-/-) not TRPA1 (-/-) mice were found more susceptible to S. aureus adhesion compared to WT. Accordingly, either ablation of TRPV1-expressing nerves or TRPV1 antagonism increased adhesion. Defense against S. aureus adhesion did not depend on nerve firing. Despite having no significant impact on CD11c+ or Lyz2+ cell numbers, the S. aureus challenge increased CD45+ cell counts, also dependent on TRPV1-expressing nerves, and it increased Lyz2+ cell sphericity and volume. Conclusions:Healthy corneas utilize TRPV1 to protect against S. aureus adhesion independently of sensory nerve firing. This contrasts with defense against P. aeruginosa adhesion which requires TRPA1 and nerve firing. How the differential immune cell responses to these two pathogens relate to TRP-dependent defense against adhesion remains to be determined.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause life- and sight-threatening infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that P. aeruginosa inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca2+ influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among P. aeruginosa subpopulations can contribute to disseminating intracellular bacteria in vivo in a mouse infection model. This study lays the groundwork for future investigations into how cooperation between extracellular and intracellular bacteria within the host contributes to disease progression and persistence.

Purpose: Healthy corneas resist colonization by virtually all microbes, yet contact lens wear can predispose the cornea to sight-threatening infection with Pseudomonas aeruginosa. Here, we explored how lens wear changes corneal epithelium transcriptional responses to P. aeruginosa and its impact on bacterial gene expression. Methods: Male and female C57BL/6J mice were fitted with a contact lens on one eye for 24 hours. After lens removal, corneas were immediately challenged for 4 hours with P. aeruginosa. A separate group of naïve mice was similarly challenged with bacteria. Bacteria-challenged eyes were compared to uninoculated naïve controls, as was lens wear alone. Total RNA sequencing determined corneal epithelium and bacterial gene expression. Results: Prior lens wear profoundly altered the corneal response to P. aeruginosa, including upregulated pattern recognition receptors (tlr3, nod1); downregulated lectin pathway of complement activation (masp1); amplified upregulation of tcf7, gpr55, ifi205, and wfdc2 (immune defense); and further suppression of efemp1 (corneal stromal integrity). Without lens wear, P. aeruginosa upregulated mitochondrial and ubiquinone metabolism genes. Lens wear alone upregulated axl, grn, tcf7, and gpr55 (immune defense) and downregulated Ca2+-dependent genes necab1, snx31, and npr3. P. aeruginosa exposure to prior lens wearing versus naïve corneas upregulated bacterial genes of virulence (popD), its regulation (rsmY, PA1226), and antimicrobial resistance (arnB, oprR). Conclusions: Prior lens wear impacts corneal epithelium gene expression, altering its responses to P. aeruginosa and how P. aeruginosa responds to it favoring virulence, survival, and adaptation. Impacted genes and associated networks provide avenues for research to better understand infection pathogenesis.

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturb this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitatively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored the contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a ΔpscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a ΔexsD mutant "constitutively on" for T3SS expression was traversal defective. The dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except for the effector genes (ΔpscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted (ΔpscU-LΔexoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented ΔpscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to the development of therapeutics targeting the T3SS system.IMPORTANCEWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of the mechanisms involved is derived largely from cell culture experimentation. The latter is due to the practical limitations of in vivo/in situ models and the challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by (i) leveraging the transparent properties and superficial location of the cornea, (ii) using our established method for enabling bacterial traversal susceptibility, and (iii) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.

Purpose:Contact lens wear induces corneal parainflammation involving increased immune cell numbers after 24 hours' (CD11c+, Lyz2+, γδ-T cells) and six days' (Ly6G+ cells) wear. We investigated the time course of onset and resolution of these responses. Methods:LysMcre or C57BL/6J mice were fitted with a contact lens (four to 48 hours). Contralateral eyes did not wear lenses. After lens removal, Lyz2+, MHC-II+ or Ly6G+ cells were examined by quantitative imaging. RT-qPCR determined cytokine gene expression. Results:Lens wear for 24 hours increased corneal Lyz2+ cells versus contralateral eyes approximately two-fold. Corneas remained free of visible pathology. The Lyz2+ response was not observed after four or 12 hours' wear, nor after 12 hours' wear plus 12 hours' no wear. Lens removal after 24 hours' wear further increased Lyz2+ cells (∼48% after one day), which persisted for four days, returning to baseline by seven days. Lyz2+ cells in contralateral eyes remained at baseline. MHC-II+ cells showed a similar response but without increasing after lens removal. Lens wear for 48 hours showed reduced Lyz2+ cells versus 24 hours' wear with one day discontinuation, correlating with reduced IL-1β and IL-18 gene expression. Lens wear for 24 hours did not induce Ly6G+ responses six days after removal. Conclusions:Lens-induced corneal parainflammation involving Lyz2+ cells requires 24 hours' wear but persists after lens discontinuation, requiring seven days for reversal. Lens wear for 48 hours may suppress initial Lyz2+ cell and cytokine responses. The significance of parainflammation during and after lens wear remains to be determined.

PURPOSE:Previously, using a murine model, we reported that contact lens (CL) wear induced corneal parainflammation involving CD11c+ cells after 24 h and Ly6G+ cells (neutrophils) after 5-6 days. Here, we investigated the role of IL-17 and γδ T cells in the CL-induced neutrophil response.METHODS:CL-wearing C57BL/6 wild-type (WT) mice were compared to lens-wearing IL-17A/F single or double gene knock-out mice, or mice treated with UC7-13D5 monoclonal antibody to functionally deplete γδ T cells. Contralateral eyes served as no lens wear controls. Corneal Ly6G+ and γδ T cell responses were quantified as was expression of genes encoding pro-inflammatory cytokines IL-17A/F, IL-β, IL-18 and expression of IL-17A/F protein.RESULTS:After 6 days lens wear, WT corneas showed Ly6G+ cell infiltration while remaining free of visible pathology. In contrast, lens-wearing corneas of IL-17AF (-/-), IL-17A (-/-) mice and γδ T cell-depleted mice showed little or no Ly6G+ cell infiltration. No Ly6G+ cell infiltration was detected in contralateral eye controls. Lens-wearing WT corneas also showed a significant increase in γδ T cells after 24 h that was maintained after 6 days of wear, and significantly increased cytokine gene expression after 6 days versus contralateral controls: IL-18 & IL-17A (∼3.9 fold) and IL-23 (∼6.5-fold). Increased IL-17A protein (∼4-fold) was detected after 6 days lens wear. γδ T cell-depletion abrogated these lens-induced changes in cytokine gene and protein expression.CONCLUSION:Together, these data show that IL-17A and γδ T cells are required for Ly6G+ cell (neutrophil) infiltration of the cornea during contact lens-induced parainflammation.

ABSTRACT Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Purpose: Contact lens wear can induce corneal parainflammation involving CD11c+ cell responses (24 hours), γδ T cell responses (24 hours and 6 days), and IL-17-dependent Ly6G+ cell responses (6 days). Topical antibiotics blocked these CD11c+ responses. Because corneal CD11c+ responses to bacteria require transient receptor potential (TRP) ion-channels (TRPA1/TRPV1), we determined if these channels mediate lens-induced corneal parainflammation. Methods: Wild-type mice were fitted with contact lenses for 24 hours or 6 days and compared to lens wearing TRPA1 (−/−) or TRPV1 (−/−) mice or resiniferatoxin (RTX)-treated mice. Contralateral eyes were not fitted with lenses. Corneas were examined for major histocompatibility complex (MHC) class II+, CD45+, γδ T, or TNF-α+ cell responses (24 hours) or Ly6G+ responses (6 days) by quantitative imaging. The quantitative PCR (qPCR) determined cytokine gene expression. Results: Lens-induced increases in MHC class II+ cells after 24 hours were abrogated in TRPV1 (−/−) but not TRPA1 (−/−) mice. Increases in CD45+ cells were unaffected. Increases in γδ T cells after 24 hours of wear were abrogated in TRPA1 (−/−) and TRPV1 (−/−) mice, as were 6 day Ly6G+ cell responses. Contralateral corneas of TRPA1 (−/−) and TRPV1 (−/−) mice showed reduced MHC class II+ and γδ T cells at 24 hours. RTX inhibited lens-induced parainflammatory phenotypes (24 hours and 6 days), blocked lens-induced TNF-α and IL-18 gene expression, TNF-α+ cell infiltration (24 hours), and reduced baseline MHC class II+ cells. Conclusions: TRPA1 and TRPV1 mediate contact lens-induced corneal parainflammation after 24 hours and 6 days of wear and can modulate baseline levels of resident corneal immune cells.

Purpose: Previously, using a murine model, we reported that contact lens (CL) wear induced corneal para-inflammation involving CD11c+ cells after 24 h and Ly6G+ cells (neutrophils) after 5-6 days. Here, we inves-tigated the role of IL-17 and gamma 8 T cells in the CL-induced neutrophil response.Methods: CL-wearing C57BL/6 wild-type (WT) mice were compared to lens-wearing IL-17A/F single or double gene knock-out mice, or mice treated with UC7-13D5 monoclonal antibody to functionally deplete gamma 8 T cells. Contralateral eyes served as no lens wear controls. Corneal Ly6G+ and gamma 8 T cell responses were quantified as was expression of genes encoding pro-inflammatory cytokines IL-17A/F, IL-(i, IL-18 and expression of IL-17A/F protein.Results: After 6 days lens wear, WT corneas showed Ly6G+ cell infiltration while remaining free of visible pa-thology. In contrast, lens-wearing corneas of IL-17AF (-/-), IL-17A (-/-) mice and gamma 8 T cell-depleted mice showed little or no Ly6G+ cell infiltration. No Ly6G+ cell infiltration was detected in contralateral eye controls. Lens-wearing WT corneas also showed a significant increase in gamma 8 T cells after 24 h that was maintained after 6 days of wear, and significantly increased cytokine gene expression after 6 days versus contralateral controls: IL-18 & IL-17A (-3.9 fold) and IL-23 (-6.5-fold). Increased IL-17A protein (-4-fold) was detected after 6 days lens wear. gamma 8 T cell-depletion abrogated these lens-induced changes in cytokine gene and protein expression.Conclusion: Together, these data show that IL-17A and gamma 8 T cells are required for Ly6G+ cell (neutrophil) infiltration of the cornea during contact lens-induced parainflammation.

Previously we reported contact lens-induced CD11c+ cell responses in healthy mouse corneas, a phenomenon that also occurs in humans. To test involvement of ocular-associated bacteria, the impact of topical antibiotics on corneal CD11c+ cell populations during 24 h of lens wear was examined. Corneas were treated with gentamicin and ofloxacin (0.3%) or gentamicin alone, some also treated prior to lens wear (24 h). Contralateral PBS-treated eyes served as controls. CD11c-YFP (Yellow Fluorescent Protein) mice allowed CD11c+ cell visualization. Viable bacteria, on the ocular surface or contact lens, were labeled using FISH (16S rRNA-targeted probe) or click-chemistry (alkDala). Antibiotic treatment reduced baseline CD11c+ cell numbers without lens wear and suppressed CD11c+ cell responses to lens wear if corneas were both pretreated and treated during wear. Few bacteria colonized corneas or lenses under any circumstances. Conjunctival commensals were significantly reduced by antibiotics with or without lens wear, but minimally impacted by lens wear alone. Deliberate inoculation with conjunctival commensals triggered CD11c+ cell responses irrespective of antibiotic pretreatment. These results suggest that while lens wear does not necessarily increase quantifiable numbers of conjunctival commensals, those neutralized by antibiotics play a role in lens-associated CD11c+ cell responses and maintaining baseline CD11c+ cell populations.

Abstract Previously we reported contact lens-induced CD11c + cell responses in healthy mouse corneas, a phenomenon that also occurs in humans. To test involvement of ocular-associated bacteria, we examined the impact of topical antibiotics on corneal CD11c + cell populations during 24 h of lens wear. Corneas were treated with gentamicin and ofloxacin (0.3%) or gentamicin alone, some also treated prior to lens wear (24 h). Contralateral PBS-treated eyes served as controls. CD11c-YFP mice allowed CD11c + cell visualization. Viable ocular- or lens-associated bacteria were labeled using FISH (16S rRNA-targeted probe) or click-chemistry (alkDala). Antibiotic treatment reduced baseline CD11c + cell numbers without lens wear and suppressed CD11c + cell responses to lens wear if corneas were both pretreated and treated during wear. Few bacteria colonized corneas or lenses under any circumstances. Conjunctival commensals were significantly reduced by antibiotics with or without lens wear, but minimally impacted by lens wear alone. Deliberate inoculation with conjunctival commensals triggered CD11c + cell responses irrespective of antibiotic pretreatment. These results suggest that while lens wear does not necessarily increase quantifiable numbers of conjunctival commensals, those neutralized by antibiotics play a role in lens-associated CD11c + cell responses and maintaining baseline CD11c + cell populations.