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Primary human corneal fibroblasts release specific matrix metalloproteinases in response to live Pseudomonas aeruginosa infection in-vitro

Primary human corneal fibroblasts release specific matrix metalloproteinases in response to live Pseudomonas aeruginosa infection in-vitro
Primary human corneal fibroblasts release specific matrix metalloproteinases in response to live Pseudomonas aeruginosa infection in-vitro
Purpose: To test the hypothesis that human corneal fibroblasts (CF) release specific matrix metalloproteinases (MMP) in respone to infection by live Pseudomonas aeruginosa bacteria.

Methods: Human CF were extracted from clinical samples, cultured to confluence in vitro and challenged with live Pseudomonas aeruginosa PA01 bacteria at an infecting dose of 107 cfu/ml as well as heat killed (HK) and freeze-thaw killed (FT) bacteria of the same dose, and lipopolysaccharides (LPS) at a dose of 100 ng/ml. Gentamicin was added to a group of cells after 3h of infection to kill all extra cellular bacteria and assess the role of internalized bacteria. Plain medium, bacteria alone (at a dose of 107 cfu/ml), and uninfected cells were used as controls. Supernatants were collected at 24h and analysed by gelatine zymography.

Results: Human CF challenged with HK bacteria, FT bacteria and LPS only produced the constitutive 65 kDa gelatinase (inactive MMP-2), similar to unchallenged cells. The same was true for CF treated with gentamicin after 3h of infection. Bacteria alone produced a 54 kDa gelatinase (alkaline protease). Human CF challenged with live bacteria released additional gelatinases of smaller sizes (25-37 kDa) that were not produced constitutively or in association with challenge with bacterial endotoxins or killed whole bacteria.

Conclusions: Human CF release specific MMPs in response to infection with live PA01 bacteria. This is probably related to exotoxins actively produced by live invading bacteria rather than passive bacterial endotoxins or structural components.
0146-0404
Elsahn, Ahmad
db5d3c4e-61a6-4069-8a20-64b077127ca6
Rodas, Paula
f74e9acd-7b1f-4527-a382-de29792c1d8f
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Hossain, Parwez
563de5fc-84ad-4539-9228-bde0237eaf51
Elsahn, Ahmad
db5d3c4e-61a6-4069-8a20-64b077127ca6
Rodas, Paula
f74e9acd-7b1f-4527-a382-de29792c1d8f
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Hossain, Parwez
563de5fc-84ad-4539-9228-bde0237eaf51

Elsahn, Ahmad, Rodas, Paula, Christodoulides, Myron and Hossain, Parwez (2014) Primary human corneal fibroblasts release specific matrix metalloproteinases in response to live Pseudomonas aeruginosa infection in-vitro. Investigative Ophthalmology & Visual Science, 55 (13).

Record type: Meeting abstract

Abstract

Purpose: To test the hypothesis that human corneal fibroblasts (CF) release specific matrix metalloproteinases (MMP) in respone to infection by live Pseudomonas aeruginosa bacteria.

Methods: Human CF were extracted from clinical samples, cultured to confluence in vitro and challenged with live Pseudomonas aeruginosa PA01 bacteria at an infecting dose of 107 cfu/ml as well as heat killed (HK) and freeze-thaw killed (FT) bacteria of the same dose, and lipopolysaccharides (LPS) at a dose of 100 ng/ml. Gentamicin was added to a group of cells after 3h of infection to kill all extra cellular bacteria and assess the role of internalized bacteria. Plain medium, bacteria alone (at a dose of 107 cfu/ml), and uninfected cells were used as controls. Supernatants were collected at 24h and analysed by gelatine zymography.

Results: Human CF challenged with HK bacteria, FT bacteria and LPS only produced the constitutive 65 kDa gelatinase (inactive MMP-2), similar to unchallenged cells. The same was true for CF treated with gentamicin after 3h of infection. Bacteria alone produced a 54 kDa gelatinase (alkaline protease). Human CF challenged with live bacteria released additional gelatinases of smaller sizes (25-37 kDa) that were not produced constitutively or in association with challenge with bacterial endotoxins or killed whole bacteria.

Conclusions: Human CF release specific MMPs in response to infection with live PA01 bacteria. This is probably related to exotoxins actively produced by live invading bacteria rather than passive bacterial endotoxins or structural components.

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More information

Published date: April 2014

Identifiers

Local EPrints ID: 448090
URI: http://eprints.soton.ac.uk/id/eprint/448090
ISSN: 0146-0404
PURE UUID: b3197614-96e2-4df0-84c8-a797941f71e1
ORCID for Myron Christodoulides: ORCID iD orcid.org/0000-0002-9663-4731
ORCID for Parwez Hossain: ORCID iD orcid.org/0000-0002-3131-2395

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Date deposited: 01 Apr 2021 15:58
Last modified: 13 Apr 2021 01:41

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