Microbicidal Effects of α- and θ-Defensins Against Antibiotic-Resistant \u3cem\u3eStaphylococcus aureus\u3c/em\u3e and \u3cem\u3ePseudomonas aeruginosa\u3c/em\u3e

Antibiotic-resistant bacterial pathogens threaten public health. Because many antibiotics target specific bacterial enzymes or reactions, corresponding genes may mutate under selection and lead to antibiotic resistance. Accordingly, antimicrobials that selectively target overall microbial cell integrity may offer alternative approaches to therapeutic design. Naturally occurring mammalian α- and θ-defensins are potent, non-toxic microbicides that may be useful for treating infections by antibiotic-resistant pathogens because certain defensin peptides disrupt bacterial, but not mammalian, cell membranes. To test this concept, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including vancomycin heteroresistant strains, and ciprofloxacin-resistant Pseudomonas aeruginosa (CipR-PA) were tested for sensitivity to α-defensins Crp-4, RMAD-4 and HNPs 1-3, and to RTD-1, macaque θ-defensin-1. In vitro, 3 μM Crp-4, RMAD-4 and RTD-1 reduced MRSA cell survival by 99%, regardless of vancomycin susceptibility. For PA clinical isolates that differ in fluoroquinolone resistance and virulence phenotype, peptide efficacy was independent of strain ciprofloxacin resistance, site of isolation or virulence factor expression. Thus, Crp-4, RMAD-4 and RTD-1 are effective in vitro antimicrobials against clinical isolates of MRSA and CipR-PA, perhaps providing templates for development of α- and θ-defensin-based microbicides against antibiotic resistant or virulent infectious agents

Retinoic acid can exacerbate T cell intrinsic TLR2 activation to promote tolerance.

The contribution of vitamin A to immune health has been well established. However, recent evidence indicates that its active metabolite, retinoic acid (RA), has the ability to promote both tolerogenic and inflammatory responses. While the outcome of RA-mediated immunity is dependent upon the immunological status of the tissue, the contribution of specific innate signals influencing this response have yet to be delineated. Here, we found that treatment with RA can dampen inflammation during intestinal injury. Importantly, we report a novel and unexpected requirement for TLR2 in RA-mediated suppression. Our data demonstrate that RA treatment enhances TLR2-dependent IL-10 production from T cells and this, in turn, potentiates T regulatory cell (TREG) generation without the need for activation of antigen presenting cells. These data also suggest that combinatorial therapy using RA and TLR2 ligands may be advantageous in the design of therapies to treat autoimmune or inflammatory disease

RA treatment suppresses IFN-γ during DSS in WT mice but enhances their secretion in TLR2KO mice.

<p>(A) Mucosal scrapings from mice receiving the DSS and water were harvested on day 10 and analyzed by ELISA for cytokine levels. (B) Quantitative RT-PCR for transcription factors associated with T helper subsets were also performed on colonic lamina propria samples taken at day 14. Data shown are the fold increase of Vehicle- and RA-treated mice compared to naive controls. Data are the mean ± SEM of 5–8 mice per group pooled from two independent experiments. (C) Expression of Foxp3 and IL-10 in colonic LP CD4⁺ T cells (n = 3 per group), one representative facs plot is shown. *, p < 0.05, **, p < 0.01 using Student’s t-test.</p

RA potentiates cytokine responses in both WT and TLR2KO DC.

<p>(A) WT splenic DC were cultured with 100 ng/ml of TLR2 ligand Pam2CysK4 and (B) WT and TLR2KO splenic DC were cultured with 10 μg/ml cecal lysate in the presence or absence of RA, TGF- β or RA/TGF- β. Supernatants were analyzed after 24 hours for cytokine production by ELISA. (C) Comparison of IL-10 IL- production from CD103⁺ LP DC and CD103⁺ SP DC. (D) Transcript levels of <i>aldh1a2</i> in spleen and LP of naïve WT and TLR2KO mice determined by quantitative PCR. For (A-C), data are the mean ± SEM of three independent experiments, for (D) 5 individual mice were analyzed. *, p < 0.05, **, p < 0.01 using Student’s t-test.</p

RA and TLR2 signals suppress pro-inflammatory cytokines from T cells.

<p>Cytokine production from purified splenic CD4⁺ T cells stimulated with anti-CD3ε and (A) Pam2CysK4, (B) cecal lysate, or (C) indicated TLR agonists in the presence or absence RA, TGF- or RA/TGF- β. Data are the mean ± SEM of 3–4 independent experiments. *, p < 0.05, **, p < 0.01 using Student’s t-test.</p

RA potentiates colonic injury and inflammation in the absence of TLR2 signaling.

<p>WT and TLR2KO mice were given 2.5% DSS for seven days to induce colonic damage and then placed on normal drinking water for seven days to allow for tissue repair. Upon first presence of fecal occult blood positivity for each, mice were supplemented with RA or vehicle control, which continued for the rest of the disease course. Weight loss (A) and fecal occult blood (B) (n = 10–14 mice per group) were monitored daily. (C) H&E staining of colonic sections of mice at day 14 (after 7 days of DSS followed by 7 days of water). (D) Histology scoring of H&E-stained colonic slides (n = 4 mice per group). (E) The percent of surface area containing ulcerated/denuded epithelium quantified using J Image software. (F) Colon lengths from mice at day 10 (n = 5–6 mice per group). *, <i>p</i> < 0.05, **, p < 0.01 using Students t-test.</p

Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa

Antibiotic-resistant bacterial pathogens threaten public health. Because many antibiotics target specific bacterial enzymes or reactions, corresponding genes may mutate under selection and lead to antibiotic resistance. Accordingly, antimicrobials that selectively target overall microbial cell integrity may offer alternative approaches to therapeutic design. Naturally occurring mammalian α- and θ-defensins are potent, non-toxic microbicides that may be useful for treating infections by antibiotic-resistant pathogens because certain defensin peptides disrupt bacterial, but not mammalian, cell membranes. To test this concept, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including vancomycin heteroresistant strains, and ciprofloxacin-resistant Pseudomonas aeruginosa (Cip(R)-PA) were tested for sensitivity to α-defensins Crp-4, RMAD-4 and HNPs 1-3, and to RTD-1, macaque θ-defensin-1. In vitro, 3 μM Crp-4, RMAD-4 and RTD-1 reduced MRSA cell survival by 99%, regardless of vancomycin susceptibility. For PA clinical isolates that differ in fluoroquinolone resistance and virulence phenotype, peptide efficacy was independent of strain ciprofloxacin resistance, site of isolation or virulence factor expression. Thus, Crp-4, RMAD-4 and RTD-1 are effective in vitro antimicrobials against clinical isolates of MRSA and Cip(R)-PA, perhaps providing templates for development of α- and θ-defensin-based microbicides against antibiotic resistant or virulent infectious agents

Genetic and Metabolic Signals during Acute Enteric Bacterial Infection Alter the Microbiota and Drive Progression to Chronic Inflammatory Disease

Chronic inflammatory disorders are thought to arise due to an interplay between predisposing host genetics and environmental factors. For example, the onset of inflammatory bowel disease is associated with enteric proteobacterial infection, yet the mechanistic basis for this association is unclear. We have shown previously that genetic defiency in TLR1 promotes acute enteric infection by the proteobacteria Yersinia enterocolitica. Examining that model further, we uncovered an altered cellular immune response that promotes the recruitment of neutrophils which in turn increases metabolism of the respiratory electron acceptor tetrathionate by Yersinia. These events drive permanent alterations in anti-commensal immunity, microbiota composition, and chronic inflammation, which persist long after Yersinia clearence. Deletion of the bacterial genes involved in tetrathionate respiration or treatment using targeted probiotics could prevent microbiota alterations and inflammation. Thus, acute infection can drive long term immune and microbiota alterations leading to chronic inflammatory disease in genetically predisposed individuals