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Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in Pglyrp-2, -3 and -4.

Gowda RN, Redfern R, Frikeche J, Pinglay S, Foster JW, Lema C, Cope L, Chakravarti S - PLoS ONE (2015)

Bottom Line: Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance.Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance.Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

ABSTRACT

Purpose: Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for Pglyrp-1 in Pseudomonas aeruginosa keratitis. Here we investigated functions of three other related genes Pglyrp-2, -3 and -4 in a mouse model of P. aeruginosa keratitis.

Methods: Wild type (WT) and each of the Pglyrp- genotypes were challenged with P. aeruginosa keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of Pglyrp-1-4, mouse beta defensins (mBD)-2,-3, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the Pglyrp- mouse types.

Results: The Pglyrp-2-/- mice showed reduced disease and lower induction of pro-inflammatory TNF-α (p = 0.02) than WT or the other Pglyrp mice. Viable bacterial yield was significantly lower in the Pglyrp-2-/- (p = 0.0007) and the Pglyrp-4-/- (p = 0.098) mice. With regards to expression of these antimicrobial genes, Pglyrp-2 expression was induced after infection in WT mice. Pglyrp-3 expression was low before and after infection in WT mice, while Pglyrp-4 expression was slightly elevated after infection in WT, Pglyrp-2 and -3 mice. Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance.

Conclusions: Efficient resolution of keratitis in the Pglyrp-2-/- mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

No MeSH data available.


Related in: MedlinePlus

Lower pro-inflammatory cytokine responses in the Pglyrp-2-/- infected eyes.Cytokines in whole eye homogenates were measured 48 h. p. i. using a cytometric bead array assay. The results show individual animals from a total of 2–3 trials. Induction of TNF-α was significantly lower (t = -2.4, p = 0.02) in Pglyrp-2-/- infected eyes. Median values for MCP-1 and IL-6 were also lower in Pglyrp-2-/- mice. Median values for all three cytokines were low in the Pglyrp-4-/- infected eyes, but the mean values did not follow this trend.
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pone.0137129.g004: Lower pro-inflammatory cytokine responses in the Pglyrp-2-/- infected eyes.Cytokines in whole eye homogenates were measured 48 h. p. i. using a cytometric bead array assay. The results show individual animals from a total of 2–3 trials. Induction of TNF-α was significantly lower (t = -2.4, p = 0.02) in Pglyrp-2-/- infected eyes. Median values for MCP-1 and IL-6 were also lower in Pglyrp-2-/- mice. Median values for all three cytokines were low in the Pglyrp-4-/- infected eyes, but the mean values did not follow this trend.

Mentions: To determine whether differential cytokine production played a role in the resolution of keratitis in the Pglyrp- mice, we measured a panel of cytokines in whole eye homogenates using a cytometric bead array assay. The cytokines assayed by this kit include IL-12p70, IFN-γ, IL-10, TNF-α, MCP-1 and IL-6. Of these, IL-12p70, IFN-γ and IL-10 could not be detected in infected or un-infected eyes of WT or of the Pglyrp- mouse genotypes. TNF-α, MCP-1 and IL-6 were undetectable in the un-infected eyes (data not shown) and induced in the infected eyes of all genotypes. Induction of TNF-α was significantly lower in infected Pglyrp-2-/- eyes compared to WT (t = -2.4, p-value = 0.02), and while IL-6 level was also low, it was not statistically significant due to an outlier. Levels of all three cytokines were also relatively low in the Pglyrp-4-/- mice without being statistically significant (Fig 4).


Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in Pglyrp-2, -3 and -4.

Gowda RN, Redfern R, Frikeche J, Pinglay S, Foster JW, Lema C, Cope L, Chakravarti S - PLoS ONE (2015)

Lower pro-inflammatory cytokine responses in the Pglyrp-2-/- infected eyes.Cytokines in whole eye homogenates were measured 48 h. p. i. using a cytometric bead array assay. The results show individual animals from a total of 2–3 trials. Induction of TNF-α was significantly lower (t = -2.4, p = 0.02) in Pglyrp-2-/- infected eyes. Median values for MCP-1 and IL-6 were also lower in Pglyrp-2-/- mice. Median values for all three cytokines were low in the Pglyrp-4-/- infected eyes, but the mean values did not follow this trend.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4558058&req=5

pone.0137129.g004: Lower pro-inflammatory cytokine responses in the Pglyrp-2-/- infected eyes.Cytokines in whole eye homogenates were measured 48 h. p. i. using a cytometric bead array assay. The results show individual animals from a total of 2–3 trials. Induction of TNF-α was significantly lower (t = -2.4, p = 0.02) in Pglyrp-2-/- infected eyes. Median values for MCP-1 and IL-6 were also lower in Pglyrp-2-/- mice. Median values for all three cytokines were low in the Pglyrp-4-/- infected eyes, but the mean values did not follow this trend.
Mentions: To determine whether differential cytokine production played a role in the resolution of keratitis in the Pglyrp- mice, we measured a panel of cytokines in whole eye homogenates using a cytometric bead array assay. The cytokines assayed by this kit include IL-12p70, IFN-γ, IL-10, TNF-α, MCP-1 and IL-6. Of these, IL-12p70, IFN-γ and IL-10 could not be detected in infected or un-infected eyes of WT or of the Pglyrp- mouse genotypes. TNF-α, MCP-1 and IL-6 were undetectable in the un-infected eyes (data not shown) and induced in the infected eyes of all genotypes. Induction of TNF-α was significantly lower in infected Pglyrp-2-/- eyes compared to WT (t = -2.4, p-value = 0.02), and while IL-6 level was also low, it was not statistically significant due to an outlier. Levels of all three cytokines were also relatively low in the Pglyrp-4-/- mice without being statistically significant (Fig 4).

Bottom Line: Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance.Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance.Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

ABSTRACT

Purpose: Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for Pglyrp-1 in Pseudomonas aeruginosa keratitis. Here we investigated functions of three other related genes Pglyrp-2, -3 and -4 in a mouse model of P. aeruginosa keratitis.

Methods: Wild type (WT) and each of the Pglyrp- genotypes were challenged with P. aeruginosa keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of Pglyrp-1-4, mouse beta defensins (mBD)-2,-3, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the Pglyrp- mouse types.

Results: The Pglyrp-2-/- mice showed reduced disease and lower induction of pro-inflammatory TNF-α (p = 0.02) than WT or the other Pglyrp mice. Viable bacterial yield was significantly lower in the Pglyrp-2-/- (p = 0.0007) and the Pglyrp-4-/- (p = 0.098) mice. With regards to expression of these antimicrobial genes, Pglyrp-2 expression was induced after infection in WT mice. Pglyrp-3 expression was low before and after infection in WT mice, while Pglyrp-4 expression was slightly elevated after infection in WT, Pglyrp-2 and -3 mice. Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance.

Conclusions: Efficient resolution of keratitis in the Pglyrp-2-/- mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

No MeSH data available.


Related in: MedlinePlus