Linezolid has unique immunomodulatory effects in post-influenza community acquired MRSA pneumonia.

INTRODUCTION:Post influenza pneumonia is a leading cause of mortality and morbidity, with mortality rates approaching 60% when bacterial infections are secondary to multi-drug resistant (MDR) pathogens. Staphylococcus aureus, in particular community acquired MRSA (cMRSA), has emerged as a leading cause of post influenza pneumonia. HYPOTHESIS:Linezolid (LZD) prevents acute lung injury in murine model of post influenza bacterial pneumonia. METHODS:Mice were infected with HINI strain of influenza and then challenged with cMRSA at day 7, treated with antibiotics (LZD or Vanco) or vehicle 6 hours post bacterial challenge and lungs and bronchoalveolar lavage fluid (BAL) harvested at 24 hours for bacterial clearance, inflammatory cell influx, cytokine/chemokine analysis and assessment of lung injury. RESULTS:Mice treated with LZD or Vanco had lower bacterial burden in the lung and no systemic dissemination, as compared to the control (no antibiotic) group at 24 hours post bacterial challenge. As compared to animals receiving Vanco, LZD group had significantly lower numbers of neutrophils in the BAL (9×10(3) vs. 2.3×10(4), p < 0.01), which was associated with reduced levels of chemotactic chemokines and inflammatory cytokines KC, MIP-2, IFN-γ, TNF-α and IL-1β in the BAL. Interestingly, LZD treatment also protected mice from lung injury, as assessed by albumin concentration in the BAL post treatment with H1N1 and cMRSA when compared to vanco treatment. Moreover, treatment with LZD was associated with significantly lower levels of PVL toxin in lungs. CONCLUSION:Linezolid has unique immunomodulatory effects on host inflammatory response and lung injury in a murine model of post-viral cMRSA pneumonia

Influenza-induced immune suppression to methicillin-resistant Staphylococcus aureus is mediated by TLR9.

Bacterial lung infections, particularly with methicillin-resistant Staphylococcus aureus (MRSA), increase mortality following influenza infection, but the mechanisms remain unclear. Here we show that expression of TLR9, a microbial DNA sensor, is increased in murine lung macrophages, dendritic cells, CD8+ T cells and epithelial cells post-influenza infection. TLR9-/- mice did not show differences in handling influenza nor MRSA infection alone. However, TLR9-/- mice have improved survival and bacterial clearance in the lung post-influenza and MRSA dual infection, with no difference in viral load during dual infection. We demonstrate that TLR9 is upregulated on macrophages even when they are not themselves infected, suggesting that TLR9 upregulation is related to soluble mediators. We rule out a role for elevations in interferon-γ (IFNγ) in mediating the beneficial MRSA clearance in TLR9-/- mice. While macrophages from WT and TLR9-/- mice show similar phagocytosis and bacterial killing to MRSA alone, following influenza infection, there is a marked upregulation of scavenger receptor A and MRSA phagocytosis as well as inducible nitric oxide synthase (Inos) and improved bacterial killing that is specific to TLR9-deficient cells. Bone marrow transplant chimera experiments and in vitro experiments using TLR9 antagonists suggest TLR9 expression on non-hematopoietic cells, rather than the macrophages themselves, is important for regulating myeloid cell function. Interestingly, improved bacterial clearance post-dual infection was restricted to MRSA, as there was no difference in the clearance of Streptococcus pneumoniae. Taken together these data show a surprising inhibitory role for TLR9 signaling in mediating clearance of MRSA that manifests following influenza infection

Mice treated with antibiotics have decreased total inflammatory cell influx as compared to saline treated animals; though LZD treated animals have lower number of neutrophils in BAL post bacterial challenge.

<p>WT mice were infected with H1N1 100 pfu i.n. and on day 7 challenged with 5×10⁷ cfu cMRSA i.t., infected mice were treated with either vehicle or antibiotics LZD (80mg/kg i.p.) or Vanco (110 mg/kg i.p.). BAL was performed 24 hours post bacterial challenge and total inflammatory cells quantitated using a hemocytometer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114574#pone.0114574.g002" target="_blank">Fig. 2A</a>) and neutrophils quantitated post cytospin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114574#pone.0114574.g002" target="_blank">Fig. 2B</a>). n = 6 in each group, experiments repeated thrice.*p < 0.05, #p < 0.01 as compared to vehicle treated group. Error bars represent mean±SEM.</p

Macrophages harvested <i>ex-vivo</i> from LZD treated mice have decreased expression of TNF-α as compared to saline treated animals post bacterial challenge.

<p>WT mice were infected with H1N1 100 pfu i.n. and on day 7 challenged with 5×10⁷ cfu cMRSA i.t., infected mice were treated with either vehicle or antibiotics LZD (80mg/kg i.p.) or Vanco (110 mg/kg i.p.). BAL was performed 24 hours post bacterial challenge, macrophages harvested by adherence purification and TNF-α gene expression measured by real-time PCR. n = 5 in each group, experiment repeated twice. *p < 0.05 as compared to vehicle treated group.</p

LZD decreased PVL toxin levels in lungs post bacterial challenge as compared to Vanco or saline treated animals.

<p>WT mice were infected with H1N1 100 pfu i.n. and on day 7 challenged with 5×10⁷ cfu cMRSA i.t., infected mice were treated with either vehicle or antibiotics LZD (80mg/kg i.p.) or Vanco (110 mg/kg i.p.). Lungs were harvested 24 hours post bacterial challenge and homogenized to single cell suspension, PVL protein levels were measured by western blot analysis and densitometry using Image J software. n = 4 in each group, experiments repeated twice. *p < 0.05, τ < 0.001 as compared to vehicle treated group.</p

Mice treated with LZD have significantly lower chemotactic chemokines as well as inflammatory cytokines in BAL post bacterial challenge.

<p>WT mice were infected with H1N1 100 pfu i.n. and on day 7 challenged with 5×10⁷ cfu cMRSA i.t., infected mice were treated with either vehicle or antibiotics LZD (80mg/kg i.p.) or Vanco (110 mg/kg i.p.). BAL was performed 24 hours post bacterial challenge and chemokine/cytokine protein levels measured by ELISA.). n = 6 in each group, experiment repeated twice. *p < 0.05, #p < 0.01 as compared to vehicle treated group. Error bars represent mean±SEM.</p

Mice treated with LZD have significantly lower lung injury as measured by protein leak as compared to Vanco or saline treated animals post bacterial challenge.

<p>WT mice were infected with H1N1 100 pfu i.n. and on day 7 challenged with 5×10⁷ cfu cMRSA i.t., infected mice were treated with either vehicle or antibiotics LZD (80mg/kg i.p.) or Vanco (110 mg/kg i.p.). Lungs were harvested 24 hours after bacterial challenge and fixed in formalin. Representative haematoxylin and eosin (H&E)-stained sections of vehicle and antibiotic treated animals 24 hours post bacterial challenge. Histology scores calculated. BAL was performed 24 hours post bacterial challenge and albumin measured by ELISA. n = 8 in each group, experiments repeated thrice. #p < 0.01, *p < 0.05 as compared to vehicle treated group. Error bars represent mean±SEM.</p

Mice infected with cMRSA post H1N1 have higher bacterial burden as compared to virus infected animals alone and treatment with antibiotics post bacterial challenge decreases bacterial burden in lung as well as protects against systemic dissemination as compared to saline treated animals.

<p>C57BL/6 mice were infected with 100pfu H1N1 i.n. and on day 7 challenged with vehicle or 5×10⁷ cMRSA, lungs and spleen harvested 24 hours post bacterial challenge and bacterial burden assessed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114574#pone.0114574.g001" target="_blank">Fig. 1A, 1B</a>). WT mice were infected with H1N1 100 pfu i.n. and on day 7 challenged with 5×10⁷ cfu cMRSA i.t., infected mice were treated with either vehicle or antibiotics LZD (80mg/kg i.p.) or Vanco (110 mg/kg i.p.). Lungs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114574#pone.0114574.g001" target="_blank">Fig. 1C</a>) and spleen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114574#pone.0114574.g001" target="_blank">Fig. 1D</a>) were harvested 24 hours post bacterial challenge and CFU quantitated. n = 6 in each group, experiments repeated thrice. #p < 0.01 as compared to vehicle treated group. Error bars represent mean±SEM.</p