Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy Improves Pseudomonas aeruginosa-Induced Keratitis.
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ABSTRACT:
SUBMITTER: Carion TW
PROVIDER: S-EPMC6210523 | biostudies-literature | 2018 Sep
REPOSITORIES: biostudies-literature
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Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy Improves <i>Pseudomonas aeruginosa</i>-Induced Keratitis.
Carion Thomas W TW Ebrahim Abdul Shukkur AS Kracht David D Agrawal Aditya A Strand Eliisa E Kaddurah Omar O McWhirter Cody R CR Sosne Gabriel G Berger Elizabeth A EA
Cells 20180920 10
With increasing multidrug resistance and contraindication for corticosteroid use, the goal of this study was to develop thymosin beta-4 (Tβ4) as an adjunctive therapy to antibiotics for the treatment of bacterial keratitis that effectively promotes enhanced wound healing, host defense, and inflammation resolution. Disease outcome was assessed by clinical score, slit lamp photography, and histopathology. Cytokine profile, bacterial load, PMN infiltration, and Griess and reactive oxygen species (R ...[more]
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Project description:Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tβ4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tβ4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tβ4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tβ4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tβ4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tβ4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study.
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Project description:The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking. We compared the relative contribution of eye and gut commensals in regulating the ocular susceptibility to Pseudomonas aeruginosa-induced keratitis. We find that in health, the presence of microbiota strengthened the ocular innate immune barrier by significantly increasing the concentrations of immune effectors in the tear film, including secretory IgA and complement proteins. Consistent with this view, Swiss Webster (SW) mice that are typically resistant to P. aeruginosa-induced keratitis become susceptible due to the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the germ free (GF) mice when compared to the conventionally maintained SW mice. The protective immunity was found to be dependent on both eye and gut microbiota with the eye microbiota having a moderate, but significant impact on the resistance to infection. These events were IL-1ß-dependent as corneal IL-1ß levels were decreased in the infected GF and antibiotic-treated mice when compared to the SPF controls, and neutralization of IL-1ß increased the ocular bacterial burden in the SPF mice. Monocolonizing GF mice with Coagulase Negative Staphylococcus sp. isolated from the conjunctival swabs was sufficient to restore resistance to infection. Cumulatively, these data underline a previously unappreciated role for microbiota in regulating susceptibility to ocular keratitis. We predict that these results will have significant implications for contact lens wearers, where alterations in the ocular commensal communities may render the ocular surface vulnerable to infections.
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Project description:PurposeContact lens wear (CLW) is one of the leading risk factors for Pseudomonas aeruginosa keratitis (PAK). However, the intrinsic factors that contribute to the high susceptibility to keratitis during CLW remain to be elucidated. CLW over an extended period can elevate corneal norepinephrine (NE) concentration. In this study, we investigated the role of NE in promoting PAK.MethodsWe constructed an injury-induced PAK model and a CLW-induced PAK model to confirm the impact of NE during corneal infection. Pharmacological blockage of NE and gene knockdown mouse were used to investigate the downstream effector of NE. RNA sequencing was performed to explore the cellular alterations during NE treatment. Non-parametric Mann-Whitney U test or Kruskal-Wallis test were used to ascertain the significance (P < 0.05).ResultsSupplementation of NE led to PAK even without artificial corneal injury during CLW. The effect was mediated by the β2-adrenergic receptor (β2-AR) in the corneal epithelium. The β2-AR blockage by the NE antagonist ICI118,551 (ICI) or by deleting of its encoding gene Adrb2 significantly alleviated infection during CLW. Conversely, β2-AR activation compromised the integrity of the epithelium and significantly increased the cortical plaque marker ezrin. Transcriptome analysis identified that the protective effect of ICI on the keratitis was mediated by dual-specificity phosphatases. Suramin, a Dusp5 antagonist, abrogated the protective effect of ICI.ConclusionsThese data reveal a new mechanism by which NE acts as an intrinsic factor that promotes CLW-induced PAK and provide novel therapeutic targets for treating keratitis by targeting NE-β2-AR.
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Project description:Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.
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Project description:BACKGROUND:Bacterial keratitis is a serious ocular infectious disease that can lead to severe visual disability. Risk factors for bacterial corneal infection include contact lens wear, ocular surface disease, corneal trauma and previous ocular or eyelid surgery. Topical antibiotics constitute the mainstay of treatment in cases of bacterial keratitis where as the use of topical corticosteroids remains controversial. Topical corticosteroids are usually used to control inflammation using the smallest amount of the drug. Their use requires optimal timing, concomitant antibiotics and careful follow up. OBJECTIVES:The objective of the review was to assess the clinical effectiveness and adverse effects of corticosteroids as adjunctive therapy for bacterial keratitis. SEARCH STRATEGY:We searched CENTRAL, MEDLINE, EMBASE, and LILACS up to 15 January 2007. We also searched the Science Citation Index to identify additional studies that had cited the included trial, an online database of ongoing trials (www.clinicaltrials.gov), reference lists of included trials, earlier reviews and the American Academy of Ophthalmology guidelines. We also contacted experts to identify any unpublished and ongoing randomized trials. SELECTION CRITERIA:We included randomized controlled trials evaluating adjunctive therapy with topical corticosteroids in people with bacterial keratitis. DATA COLLECTION AND ANALYSIS:Two review authors independently screened all the retrieved articles. Methodological quality of the one included trial was assessed using forms developed using pre-specified criteria by at least two review authors. We planned to extract data on outcomes using forms developed for the purpose. We planned to report risk ratios for dichotomous outcomes and mean differences for continuous outcomes. MAIN RESULTS:A single trial was eligible for inclusion in the review. Participants in the trial were randomized using a random numbers table. Allocation concealment was not attempted. Masking of participants, and care-providers was also not attempted. Outcome assessment was conducted independently by two physicians. Neither was masked to the treatment allocation. The trial reported the healing rate of epithelial defects and improvement in visual acuity. AUTHORS' CONCLUSIONS:There are no good quality randomized trials evaluating the effects of adjunct use of topical corticosteroids in bacterial keratitis. The only randomized trial we identified in the literature suffered from major methodological inadequacies.
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Project description:PURPOSE:Glycyrrhizin (GLY), an inhibitor of high-mobility group box 1 (HMGB1) protects prophylactically against Pseudomonas aeruginosa keratitis. However, the therapeutic potential of GLY to enhance an antibiotic has not been tested and is our purpose. METHODS:C57BL/6 mice (B6) were infected with a clinical isolate (KEI 1025) of P. aeruginosa and treated topically at 6 h postinfection (p.i.) with GLY or phosphate-buffered saline (PBS). Clinical scores, photography with a slit lamp, enzyme-linked immunosorbent assay, myeloperoxidase assay, bacterial plate counts, histopathology, reactive oxygen/nitrogen species (ROS/RNS) assays, and in vitro macrophage (Mφ) stimulation assays were used to assess effects of GLY treatment. In separate similar experiments, the ability of GLY to bioenhance the antibiotic, tobramycin (TOB), was assessed. RESULTS:In vivo, GLY versus PBS topical treatment began at 6 h p.i., improved disease outcome by significantly reducing clinical scores, proinflammatory proteins (HMGB1, RAGE, TLR4, TNF-α, and CXCL2), neutrophil infiltrate, bacterial load, ROS/RNS, and nitric oxide. In vitro, GLY downregulated iNOS and COX-2 expression (mRNA) in both mouse and human (THP-1) Mφ. At 6 and 24 h p.i., treatment with GLY enhanced the effects of TOB compared with TOB alone by significantly reducing corneal bacterial load and/or protein levels of cytokines CXCL2 and IL-1β. CONCLUSIONS:Data provide evidence that GLY is not only therapeutic for Pseudomonas keratitis through its ability to reduce HMGB1, bacterial load, and oxidative damage but also through its bioenhancement of an antibiotic, even when treatment is initiated at 24 h after infection.
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Project description:PurposeTo determine whether rapamycin altered corneal growth factor levels to impact severity of Pseudomonas aeruginosa keratitis.MethodsBALB/c mice were injected intraperitoneally with rapamycin or PBS and infected with P. aeruginosa. Corneas were harvested and mRNA levels of growth factors (EGF, HGF, FGF-7/KGF), receptors (EGFR, c-met, FGFR-2), and signaling molecules (PI3K, Akt, S6K1, and IGF-1R) tested. ELISA determined HGF/c-met, IGF-1, and Substance P (SP) protein levels. Corneal application of recombinant (r)HGF was assessed by clinical score, photography with a slit lamp, real-time RT-PCR (mRNA for mT0R, IL-10, IL-12, IL-18, PI3KCα, Akt), and ELISA (total and phosphorylated [p]c-met); rIGF-1 effects also were tested by ELISA. In vitro, RAW cells and peritoneal macrophages were stimulated with LPS ± rHGF ± c-met inhibitor (CI) and mTOR mRNA levels tested.ResultsRapamycin disparately regulated infected corneal mRNA levels of EGF/EGFR and FGF-7/FGFR-2, but HGF/c-met mRNA levels both increased. ELISA confirmed elevated HGF protein. Rapamycin did not change PI3KCα or Akt signaling molecule expression, downregulated S6K1, but upregulated IGF-1R mRNA levels; IGF-1 and SP proteins also were upregulated. After infection, topical rHGF versus PBS increased mRNA levels of IL-12p40, IL-18, PI3KCα, and Akt; mTOR and IL-10 mRNA were downregulated; rIGF-1 increased HGF protein. In vitro, rHGF and LPS lowered RAW cell and macrophage mTOR levels; CI addition restored them.ConclusionsCollectively, these data provide evidence that enhanced corneal HGF levels increase signaling through the c-met receptor, decrease mTOR levels, and enhance proinflammatory cytokines, while decreasing anti-inflammatory cytokines, and that HGF signaling is central to disease outcome.
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Project description:PURPOSE:To compare the clinical course and effect of adjunctive corticosteroid therapy in Pseudomonas aeruginosa with those of all other strains of bacterial keratitis. METHODS:Subanalyses were performed on data collected in the Steroids for Corneal Ulcers Trial (SCUT), a large randomized controlled trial in which patients were treated with moxifloxacin and were randomly assigned to 1 of 2 adjunctive treatment arms: corticosteroid or placebo (4 times a day with subsequent reduction). Multivariate analysis was used to determine the effect of predictors, organism, and treatment on outcomes, 3-month best-spectacle-corrected visual acuity (BSCVA), and infiltrate/scar size. The incidence of adverse events over a 3-month follow-up period was compared using Fisher's exact test. RESULTS:SCUT enrolled 500 patients. One hundred ten patients had P. aeruginosa ulcers; 99 of 110 (90%) enrolled patients returned for follow-up at 3 months. Patients with P. aeruginosa ulcers had significantly worse visual acuities than patients with other bacterial ulcers (P = 0.001) but showed significantly more improvement in 3-month BSCVA than those with other bacterial ulcers, adjusting for baseline characteristics (-0.14 logMAR; 95% confidence interval, -0.23 to -0.04; P = 0.004). There was no significant difference in adverse events between P. aeruginosa and other bacterial ulcers. There were no significant differences in BSCVA (P = 0.69), infiltrate/scar size (P = 0.17), and incidence of adverse events between patients with P. aeruginosa ulcers treated with adjunctive corticosteroids and patients given placebo. CONCLUSIONS:Although P. aeruginosa corneal ulcers have a more severe presentation, they appear to respond better to treatment than other bacterial ulcers. The authors did not find a significant benefit with corticosteroid treatment, but they also did not find any increase in adverse events. (ClinicalTrials.gov number, NCT00324168.).
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Project description:A worrisome trend in the study and treatment of infectious disease noted in recent years is the increase in multidrug resistant strains of bacteria concurrent with a scarcity of new antimicrobial agents to counteract this rise. This is particularly true amongst bacteria within the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) designation. P. aeruginosa is one of the most common causes of bacterial keratitis. Therefore, it is of vital importance to characterize new antimicrobial agents with anti-Pseudomonal activity for use with the ocular surface. MEDI3902 is a multifunctional antibody that targets the P. aeruginosa persistence factor Psl exopolysaccharide, and the type 3 secretion protein PcrV. We initially assessed this antibody for ocular surface toxicity. The antimicrobial activity of the antibody was then tested by treating mice with established P. aeruginosa keratitis with both topical and intravenous treatment modalities. MEDI3902, was shown to be non-toxic to the ocular surface of mice when given topically. It was also effective compared to the control antibody at preventing P. aeruginosa keratitis with a one-time treatment at the time of infection. Both topical and intravenous administration of MEDI3902 has been proved significant in treating established keratitis infections as well, speeding the resolution of infection significantly more than that of the control IgG. We report the first use of a topical immunotherapeutic multifunctional agent targeting Psl and type 3 secretion on the ocular surface as an antimicrobial agent. While MEDI3902 has been shown to prevent Pseudomonas biofilm formation in keratitis models when given prophylactically intravitally, we show that MEDI3902 has the capability to also treat an active infection when given intravenously to mice with Pseudomonas keratitis. Our data indicate antibodies are well tolerated and nontoxic on the ocular surface. They reduce infection in mice treated concurrently at inoculation and reduced the signs of cornea pathology in mice with established infection. Taken together, these data indicate treatment with monoclonal antibodies directed against Psl and PcrV may be clinically effective in the treatment of P. aeruginosa keratitis and suggest that the design of further antibodies to be an additional tool in the treatment of bacterial keratitis.
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Project description:With few novel antimicrobials in development, resistance to the current selection of antibiotics increasingly encroaches on our ability to control microbial infections. One limitation in our understanding of the basis of the constraints on current therapies is our poor understanding of antibiotic interactions with bacteria on a global scale. Custom DNA microarrays were used to characterize the response of Pseudomonas aeruginosa to ciprofloxacin, a fluoroquinolone commonly used in therapy against chronic infections by this intrinsically resistant bacterium. Of the approximately 5,300 open reading frames (ORFs) on the array, 941 genes showed statistically significant (P </= 0.05) differential expression in response to 0.3x MIC of ciprofloxacin; 554 were promoted and 387 were repressed. Most striking among the responsive genes was the region between PA0613 and PA0648, which codes for the bacteriophage-like R2/F2 pyocins. In this region, virtually every ORF was increased by 0.3x MIC of ciprofloxacin and even more dramatically up-regulated (7- to 19-fold) following treatment with 1x MIC of ciprofloxacin. Pyocin gene expression was confirmed with lux reporter mutants and real-time PCR studies; pyocin-like particles were also present in transmission electron micrographs of supernatants from cells treated with 1x MIC of ciprofloxacin. Interestingly, mutants in this region exhibited >/=8-fold-increased resistance to ciprofloxacin and other fluoroquinolones, demonstrating that this region is a susceptibility determinant. Since this region is known to be variably present in the genomes of clinical isolates of P. aeruginosa (R. K. Ernst et al., Environ. Microbiol. 5:1341-1349, 2003, and M. C. Wolfgang et al., Proc. Natl. Acad. Sci. USA 100:8484-8489, 2003), these findings demonstrate that the R2/F2 pyocin region is a "loaded gun" that can mediate fluoroquinolone susceptibility in P. aeruginosa.
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