Image_1_Integrin α2β1 Expression Regulates Matrix Metalloproteinase-1-Dependent Bronchial Epithelial Repair in Pulmonary Tuberculosis.PDF

<p>Pulmonary tuberculosis (TB) is caused by inhalation of Mycobacterium tuberculosis, which damages the bronchial epithelial barrier to establish local infection. Matrix metalloproteinase-1 plays a crucial role in the immunopathology of TB, causing breakdown of type I collagen and cavitation, but this collagenase is also potentially involved in bronchial epithelial repair. We hypothesized that the extracellular matrix (ECM) modulates M. tuberculosis-driven matrix metalloproteinase-1 expression by human bronchial epithelial cells (HBECs), regulating respiratory epithelial cell migration and repair. Medium from monocytes stimulated with M. tuberculosis induced collagenase activity in bronchial epithelial cells, which was reduced by ~87% when cells were cultured on a type I collagen matrix. Matrix metalloproteinase-1 had a focal localization, which is consistent with cell migration, and overall secretion decreased by 32% on type I collagen. There were no associated changes in the specific tissue inhibitors of metalloproteinases. Decreased matrix metalloproteinase-1 secretion was due to ligand-binding to the α2β1 integrin and was dependent on the actin cytoskeleton. In lung biopsies, samples from patients with pulmonary TB, integrin α2β1 is highly expressed on the bronchial epithelium. Areas of lung with disrupted collagen matrix showed an increase in matrix metalloproteinases-1 expression compared with areas where collagen was comparable to control lung. Type I collagen matrix increased respiratory epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which α2β1-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB.</p

Table_1_Integrin α2β1 Expression Regulates Matrix Metalloproteinase-1-Dependent Bronchial Epithelial Repair in Pulmonary Tuberculosis.PDF

<p>Pulmonary tuberculosis (TB) is caused by inhalation of Mycobacterium tuberculosis, which damages the bronchial epithelial barrier to establish local infection. Matrix metalloproteinase-1 plays a crucial role in the immunopathology of TB, causing breakdown of type I collagen and cavitation, but this collagenase is also potentially involved in bronchial epithelial repair. We hypothesized that the extracellular matrix (ECM) modulates M. tuberculosis-driven matrix metalloproteinase-1 expression by human bronchial epithelial cells (HBECs), regulating respiratory epithelial cell migration and repair. Medium from monocytes stimulated with M. tuberculosis induced collagenase activity in bronchial epithelial cells, which was reduced by ~87% when cells were cultured on a type I collagen matrix. Matrix metalloproteinase-1 had a focal localization, which is consistent with cell migration, and overall secretion decreased by 32% on type I collagen. There were no associated changes in the specific tissue inhibitors of metalloproteinases. Decreased matrix metalloproteinase-1 secretion was due to ligand-binding to the α2β1 integrin and was dependent on the actin cytoskeleton. In lung biopsies, samples from patients with pulmonary TB, integrin α2β1 is highly expressed on the bronchial epithelium. Areas of lung with disrupted collagen matrix showed an increase in matrix metalloproteinases-1 expression compared with areas where collagen was comparable to control lung. Type I collagen matrix increased respiratory epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which α2β1-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB.</p

Image_2_Integrin α2β1 Expression Regulates Matrix Metalloproteinase-1-Dependent Bronchial Epithelial Repair in Pulmonary Tuberculosis.PDF

<p>Pulmonary tuberculosis (TB) is caused by inhalation of Mycobacterium tuberculosis, which damages the bronchial epithelial barrier to establish local infection. Matrix metalloproteinase-1 plays a crucial role in the immunopathology of TB, causing breakdown of type I collagen and cavitation, but this collagenase is also potentially involved in bronchial epithelial repair. We hypothesized that the extracellular matrix (ECM) modulates M. tuberculosis-driven matrix metalloproteinase-1 expression by human bronchial epithelial cells (HBECs), regulating respiratory epithelial cell migration and repair. Medium from monocytes stimulated with M. tuberculosis induced collagenase activity in bronchial epithelial cells, which was reduced by ~87% when cells were cultured on a type I collagen matrix. Matrix metalloproteinase-1 had a focal localization, which is consistent with cell migration, and overall secretion decreased by 32% on type I collagen. There were no associated changes in the specific tissue inhibitors of metalloproteinases. Decreased matrix metalloproteinase-1 secretion was due to ligand-binding to the α2β1 integrin and was dependent on the actin cytoskeleton. In lung biopsies, samples from patients with pulmonary TB, integrin α2β1 is highly expressed on the bronchial epithelium. Areas of lung with disrupted collagen matrix showed an increase in matrix metalloproteinases-1 expression compared with areas where collagen was comparable to control lung. Type I collagen matrix increased respiratory epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which α2β1-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB.</p

Image_3_Integrin α2β1 Expression Regulates Matrix Metalloproteinase-1-Dependent Bronchial Epithelial Repair in Pulmonary Tuberculosis.PDF

<p>Pulmonary tuberculosis (TB) is caused by inhalation of Mycobacterium tuberculosis, which damages the bronchial epithelial barrier to establish local infection. Matrix metalloproteinase-1 plays a crucial role in the immunopathology of TB, causing breakdown of type I collagen and cavitation, but this collagenase is also potentially involved in bronchial epithelial repair. We hypothesized that the extracellular matrix (ECM) modulates M. tuberculosis-driven matrix metalloproteinase-1 expression by human bronchial epithelial cells (HBECs), regulating respiratory epithelial cell migration and repair. Medium from monocytes stimulated with M. tuberculosis induced collagenase activity in bronchial epithelial cells, which was reduced by ~87% when cells were cultured on a type I collagen matrix. Matrix metalloproteinase-1 had a focal localization, which is consistent with cell migration, and overall secretion decreased by 32% on type I collagen. There were no associated changes in the specific tissue inhibitors of metalloproteinases. Decreased matrix metalloproteinase-1 secretion was due to ligand-binding to the α2β1 integrin and was dependent on the actin cytoskeleton. In lung biopsies, samples from patients with pulmonary TB, integrin α2β1 is highly expressed on the bronchial epithelium. Areas of lung with disrupted collagen matrix showed an increase in matrix metalloproteinases-1 expression compared with areas where collagen was comparable to control lung. Type I collagen matrix increased respiratory epithelial cell migration in a wound-healing assay, and this too was matrix metalloproteinase-dependent, since it was blocked by the matrix metalloproteinase inhibitor GM6001. In summary, we report a novel mechanism by which α2β1-mediated signals from the ECM modulate matrix metalloproteinase-1 secretion by HBECs, regulating their migration and epithelial repair in TB.</p

<i>emm</i>3 strains have low mitogenicity due to the mutation in <i>smeZ</i>.

<p><i>A.</i> MNC proliferative response to culture supernatants from an <i>emm</i>3 isolate, GAS-M3 carrying the typical M3-<i>smeZ</i> with 13 bp deletion, GAS-M3 over-expressing the functional M89 form of SMEZ (GAS-M3<i>_smeZ</i>_-M89) and GAS-M3 over-expressing the M3-SMEZ (GAS-M3<i>_smeZ</i>_-M3). <i>B.</i> Experimental <i>smeZ</i> mutation in <i>emm</i>1 <i>S. pyogenes</i> (GAS-M1) reduced MNC proliferation (GAS-M1Δ<i>smeZ</i>). Proliferative response was restored when GAS-M1Δ<i>smeZ</i> over-expressed the functional M89 form of SMEZ (GAS-M1<i>_smeZ</i>_-M89) but not with M3-type form of SMEZ (GAS-M1<i>_smeZ</i>_-M3). <i>C</i>. A similar result was obtained using an <i>emm</i>89 strain (GAS-M89) with an experimental mutation in <i>smeZ</i> (GAS-M89Δ<i>smeZ</i>). Proliferation was restored when GAS-M89ΔsmeZ over-expressed the functional M89 form of SMEZ (GAS-M89<i>_smeZ</i>_-M89) but not with M3-type form of SMEZ (GAS-M89<i>_smeZ</i>_-M3). Negative; media alone.Proliferation was measured as counts per minute (cpm) of tritiated-thymidine uptake and the percentage proliferation for each strain was calculated relative to the wild type strain (GAS-M3, GAS-M1, GAS-M89 respectively). Data are mean (+standard deviation) of three measurements. Representative of two experiments performed using different donor MNC.</p

<i>Emm</i>3 STSS isolates show <i>emm</i>-type specific differences in overall mitogenicity.

<p><i>A.</i>Human MNC proliferation response to culture supernatants from 63 <i>Streptococcus pyogenes</i> streptococcal toxic shock syndrome (STSS) isolates grouped by <i>emm</i>-type (numbers per group: <i>emm</i>1, n = 11; <i>emm</i>3, n = 12; <i>emm1</i>2 and <i>emm</i>87, n = 11; <i>emm</i>18, n = 5; <i>emm</i>28, n = 7; <i>emm</i>89, n = 6). Negative; tissue culture media (RMPI) alone. Outliers are represented as individual circles. Representative of 2 experiments performed on different donors. <i>B.</i> Human MNC proliferation response to sera obtained from CD1 mice 24 hours after being infected with one of three <i>S. pyogenes</i> strains representing each <i>emm</i>-type; two mice were infected per strain. Negative; uninfected mouse serum. Proliferative response is measured as counts per minute (cpm) of tritiated-thymidine uptake. Median and 5th, 25th, 50th, 75th, and 95th centiles shown.</p

<i>emm</i>3 isolates have a 13 base pair (bp) deletion within the <i>smeZ</i> locus.

<p>Representation of the <i>smeZ</i> locus from <i>emm</i>1 (M1) and <i>emm</i>3 (M3) strains. Nucleotides 1 to 3 encode the start codon (shown in bold). From 73 bp of the nucleotide sequence, the amino acid sequence of the mature SMEZ protein is shown. <i>Emm</i>3 strains have a 13 bp deletion at 316 bp (highlighted by a shaded box) that results in a frameshift and a predicted premature stop codon after 86 amino acids (shown as *). The forward primer and the reverse primer amplify the full length <i>smeZ</i> locus. The 13 bp deletion was detected using a forward (truncated) primer that anneals specifically to the region containing the deletion.</p

<i>S. pyogenes</i> strains used in this study.

<p><i>S. pyogenes</i> strains used in this study.</p

Functional SMEZ is required to stimulate production of cytokines from human tonsil cells.

<p>Human tonsil cell suspensions were cultured with bacterial cell-free culture supernatants from GAS-M3_control(white bars), GAS-M3<i>_smeZ</i>_-M89 (black bars) and GAS-M3<i>_smeZ</i>_-M3 (gray bars). After 2 and 5 days incubation cell-free media were obtained from cultures and production of TNFα, TNFβ, IL-10, IL-17 and IFNγ were measured by ELISA. Horizontal dotted lines represent the mean level of cytokines produced after co-culture with bacterial cell-free culture supernatants from GAS-M1_control on day 2 and day 5. Mean (+ standard deviation) of three replicates measured in duplicate. Representative of two experiments performed on different donors. Statistical analysis was performed using ANOVA with Bonferroni multiple comparison.</p