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Suzanne Fleiszig, David Evans, Connie Tam; MyD88-dependent and -independent antimicrobial activity in mouse corneas induced by Pseudomonas aeruginosa antigen challenge. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5207.
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Previously, we reported that invasive strains of Pseudomonas aeruginosa traversed through the MyD88 knockout, but not wild-type, mouse corneal epithelium. Here, we examined corneal lysates of wild-type and MyD88 knockout mouse corneas to elucidate MyD88-dependent host defense factors involved.
Corneas of C57 BL/6 wild-type and MyD88 knockout mice were homogenized in distilled water (or Tris buffer for mass spectrometry). Corneal lysates were serially fractioned by size using centrifugal filters of a 100, 30 and 10 kDa cutoff. P. aeruginosa (10^6 cfu/ml) was incubated in lysate fractions for 3 h and bacterial viable counts were determined. Mass spectrometry was used to identify peptides and proteins in the lysate fractions.
Under normal healthy conditions, the 30-100 kDa and <10 kDa corneal lysate fractions of untreated wild-type, but not MyD88 knockout mice, retarded the growth of P. aeruginosa (P < 0.005). After exposure to P. aeruginosa antigens, the <10 kDa fractions of both wild-type and MyD88 knockout mice were anti-pseudomonal. In addition, the antibacterial activity of wild-type 30-100 kDa fraction was magnified. Mass spectrometric analysis comparing the 30-100 kDa fraction of wild-type to MyD88 knockouts showed that P. aeruginosa antigen exposure resulted in MyD88-dependent upregulation of 17 proteins (e.g. glutathione S-transferase, annexin, aspartic protease, GDP dissociation inhibitor) and downregulation of 2 proteins (fatty acid binding protein and profilin) by 2-fold or more.
For corneal factors in the 30-100 kDa range, MyD88 was found critical for both constitutive and antigen activated antimicrobial activity. For factors < 10 kDa, only constitutive antimicrobial activity was found to be MyD88 dependent. While the <10 kDa cell lysate fractions were more antimicrobial if cells had been exposed to antigens, this enhanced activity was not MyD88-dependent. These data suggest that while MyD88 plays an important role in maintaining the antimicrobial activity of corneal epithelial cells, MyD88-independent pathways can contribute when there is microbial ligand exposure involving factors less than 10 kDa.
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