Tenascin C Has a Modest Protective Effect on Acute Lung Pathology during Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia in Mice

Abstract

Tenascin C (TNC) is an extracellular matrix protein with immunomodulatory properties that plays a major role during tissue injury and repair. TNC levels are increased in patients with pneumonia and pneumosepsis, and they are associated with worse outcomes. Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium that is a major causative pathogen in nosocomial pneumonia and a rising cause of community-acquired pneumonia. To study the role of TNC during MRSA-induced pneumonia, TNC sufficient (TNC^+/+) and TNC-deficient (TNC^-/-) mice were infected with MRSA via the airways and euthanized after 6, 24, and 48 h for analysis. Pulmonary transcription of TNC peaked at 6 h, while immunohistochemistry revealed higher protein levels at later time points. Although TNC deficiency was not associated with changes in bacterial clearance, TNC^-/- mice showed increased levels of TNF-α and IL-6 in bronchoalveolar lavage fluid during the acute phase of infection when compared with TNC^+/+ mice. In addition, TNC^-/- mice showed more severe pulmonary pathology at 6, but not at 24 or 48 h, after infection. Together, these data suggest that TNC plays a moderate protective role against tissue pathology during the acute inflammatory phase, but not during the bacterial clearance phase, of MRSA-induced pneumonia. These results argue against an important role of TNC on disease outcome during MRSA-induced pneumonia. IMPORTANCE Recently, the immunomodulatory properties of TNC have drawn substantial interest. However, to date most studies made use of sterile models of inflammation. In this study, we examine the pathobiology of MRSA-induced pneumonia in a model of TNC-sufficient and TNC-deficient mice. We have studied the immune response and tissue pathology both during the initial insult and also during the resolution phase. We demonstrate that MRSA-induced pneumonia upregulates pulmonary TNC expression at the mRNA and protein levels. However, the immunomodulatory role of TNC during bacterial pneumonia is distinct from models of sterile inflammation, indicating that the function of TNC is context dependent. Contrary to previous descriptions of TNC as a proinflammatory mediator, TNC-deficient mice seem to suffer from enhanced tissue pathology during the acute phase of infection. Nonetheless, besides its role during the acute phase response, TNC does not seem to play a major role in disease outcome during MRSA-induced pneumonia.

Keywords: Gram-positive bacterial infections; Staphylococcus aureus; alarmins; immune system; innate immunity; methicillin-resistant Staphylococcus aureus; mice; pneumonia; tenascin C.

FIG 1

Tenascin C expression is induced during a transient pulmonary infection with methicillin-resistant Staphylococcus aureus. (A) Mice were intranasally infected with MRSA. RNA was extracted from the lung homogenate of mice sacrificed before infection and at 6, 24, or 48 h thereafter. TNC mRNA expression was determined in the tissue of TNC^+/+ mice through qPCR and normalized to HPRT1 mRNA content. (B) Immunohistochemistry staining of tenascin C in pulmonary tissue during infection. Pictures are representative of 5 independent samples. (C) BALF was collected upon sacrificing mice at 6, 24, and 48 h after infection. The number of CFU in BALF was determined through overnight culture on blood agar plates. Data are shown as a Tukey boxplot. TNC mRNA levels were compared to baseline using unpaired t tests with Holm’s adjustment. Bacterial outgrowth between TNC^+/+ and TNC^−/− mice after 6 h was compared using an unpaired t test. Later time points were limited to descriptive statistics without hypothesis testing. The dashed line represents the lower limit of detection. **, P < 0.01 (compared to naive).

FIG 2

Tenascin C is not required for the recruitment and activation of monocytes during pulmonary infection with methicillin-resistant Staphylococcus aureus. Mice were intranasally infected with MRSA. Before infection and at 6, 24, and 48 h thereafter, mice were sacrificed and unilateral BALF was collected. (A) Total numbers of alveolar macrophages, monocytes, inflammatory monocytes, and noninflammatory monocytes were determined through fluorescence-activated cell sorting (FAC) analysis. (B) The expression of CD11b on alveolar macrophages was measured through the median fluorescence intensity during FACS analysis. (C) Cytokine levels in BALF were determined through ELISA. Data are shown as Tukey boxplots. Statistical analysis was performed using linear models. Dashed lines indicate the lower limit of quantification. *, P < 0.05; **, P < 0.01.

FIG 3

Tenascin C plays a limited role in neutrophil recruitment and activation during methicillin-resistant Staphylococcus aureus-induced pneumonia. Mice were intranasally infected with MRSA. Before infection and at 6, 24, and 48 h thereafter, mice were sacrificed. Unilateral BALF and contralateral lung tissue was collected. (A) Tissue was fixed, embedded in paraffin, and stained for Ly-6G. Ly-6G positivity is expressed as a percentage of total tissue surface. (B) Total neutrophil content of BALF was analyzed through FACS. (C) Neutrophil CD11b surface expression levels were assessed through the median fluorescence intensity during FACS analysis. (D and E) BALF myeloperoxidase (D) and neutrophil elastase (E) levels were determined through ELISA. Data are shown as Tukey boxplots. Statistical analysis was performed using linear models.

FIG 4

Tenascin C offers limited protection against early tissue pathology during methicillin-resistant Staphylococcus aureus-induced pneumonia. Mice were infected through intranasal administration of MRSA. Mice were sacrificed at 6, 24, and 48 h thereafter and lung tissue and BALF were collected. (A) To determine total tissue pathology, tissue was fixed and embedded in paraffin. H&E-stained slides were prepared and subsequently scored for tissue pathology by a pathologist blinded for the group condition. (B) Representative H&E-stained slides of TNC^+/+ (top) and TNC^−/− mice (bottom) at 6 h after infection. (C) As a measure for integrity of the pulmonary barrier, protein leak into the alveolar space was measured as total protein content of the BALF trough a bicinchoninic acid protein assay. Data are shown as Tukey boxplots. Statistical analysis was performed using linear models. *, P < 0.05.