Matrix metalloproteinases mediate influenza A-associated shedding of the alveolar epithelial glycocalyx.

BACKGROUND: The alveolar epithelium is protected by a heparan sulfate-rich, glycosaminoglycan layer called the epithelial glycocalyx. It is cleaved in patients with acute respiratory distress syndrome (ARDS) and in murine models of influenza A (IAV) infection, shedding fragments into the airspace from the cell surface. Glycocalyx shedding results in increased permeability of the alveolar-capillary barrier, amplifying acute lung injury. The mechanisms underlying alveolar epithelial glycocalyx shedding in IAV infection are unknown. We hypothesized that induction of host sheddases such as matrix metalloproteinases (MMPs) during IAV infection results in glycocalyx shedding and increased lung injury. MATERIALS AND METHODS: We measured glycocalyx shedding and lung injury during IAV infection with and without treatment with the pan-MMP inhibitor Ilomastat (ILO) and in an MMP-7 knock out (MMP-7KO) mouse. C57BL/6 or MMP-7KO male and female mice were given IAV A/PR/8/34 (H1N1) at 30,000 PFU/mouse or PBS intratracheally. For some experiments, C56BL/6 mice were infected in the presence of ILO (100mg/kg) or vehicle given daily by IP injection. Bronchoalveolar lavage (BAL) and lung tissue were collected on day 1, 3, and 7 for analysis of glycocalyx shedding (BAL Syndecan-1) and lung injury (histology, BAL protein, BAL cytokines, BAL immune cell infiltrates, BAL RAGE). Expression and localization of the sheddase MMP-7 and its inhibitor TIMP-1 was examined by RNAScope. For in vitro experiments, MLE-12 mouse lung epithelial cells were cultured and treated with active or heat-inactivated heparinase (2.5 U/mL) prior to infection with IAV (MOI 1) and viral load and MMP-7 and TIMP-1 expression analyzed. RESULTS: IAV infection caused shedding of the epithelial glycocalyx into the BAL. Inhibition of MMPs with ILO reduced glycocalyx shedding by 36% (p = 0.0051) and reduced lung epithelial injury by 40% (p = 0.0404). ILO also reduced viral load by 68% (p = 0.027), despite having no significant effect on lung cytokine production. Both MMP-7 and its inhibitor TIMP-1 were upregulated in IAV infected mice: MMP-7 colocalized with IAV, while TIMP-1 was limited to cells adjacent to infection. However, MMP-7KO mice had similar glycocalyx shedding, epithelial injury, and viral load compared to WT littermates, suggesting redundancy in MMP sheddase function in the lung. In vitro, heparinase treatment before infection led to a 52% increase in viral load (p = 0.0038) without altering MMP-7 or TIMP-1 protein levels. CONCLUSIONS: Glycocalyx shedding and MMPs play key roles in IAV-induced epithelial injury, with significant impact on IAV viral load. Further studies are needed to understand which specific MMPs regulate lung epithelial glycocalyx shedding.

Cell-free hemoglobin triggers macrophage cytokine production via TLR4 and MyD88.

Cell-free hemoglobin (CFH) is elevated in the airspace of patients with acute respiratory distress syndrome (ARDS) and is sufficient to cause acute lung injury in a murine model. However, the pathways through which CFH causes lung injury are not well understood. Toll-like receptor 4 (TLR4) is a mediator of inflammation after detection of damage- and pathogen-associated molecular patterns. We hypothesized that TLR4 signaling mediates the proinflammatory effects of CFH in the airspace. After intratracheal CFH, BALBc mice deficient in TLR4 had reduced inflammatory cell influx into the airspace [bronchoalveolar lavage (BAL) cell counts, median TLR4 knockout (KO): 0.8 × 104/mL [IQR 0.4-1.2 × 104/mL], wild-type (WT): 3.0 × 104/mL [2.2-4.0 × 104/mL], P < 0.001] and attenuated lung permeability (BAL protein, TLR4KO: 289 µg/mL [236-320], WT: 488 µg/mL [422-536], P < 0.001). These mice also had attenuated production of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the airspace. C57Bl/6 mice lacking TLR4 on myeloid cells only (LysM.Cre+/-TLR4fl/fl) had reduced cytokine production in the airspace after CFH, without attenuation of lung permeability. In vitro studies confirm that WT primary murine alveolar macrophages exposed to CFH (0.01-1 mg/mL) had dose-dependent increases in IL-6, IL-1 ß, CXC motif chemokine ligand 1 (CXCL-1), TNF-α, and IL-10 (P < 0.001). Murine MH-S alveolar-like macrophages show TLR4-dependent expression of IL-1ß, IL-6, and CXCL-1 in response to CFH. Primary alveolar macrophages from mice lacking TLR4 adaptor proteins myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-ß (TRIF) revealed that MyD88KO macrophages had 71-96% reduction in CFH-dependent proinflammatory cytokine production (P < 0.001), whereas macrophages from TRIFKO mice had variable changes in cytokine responses. These data demonstrate that myeloid TLR4 signaling through MyD88 is a key regulator of airspace inflammation in response to CFH.NEW & NOTEWORTHY Cell-free hemoglobin (CFH) is elevated in the airspace of most patients with acute respiratory distress syndrome and causes severe inflammation. Here, we identify that CFH contributes to macrophage-induced cytokine production via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling. These data increase our knowledge of the mechanisms through which CFH contributes to lung injury and may inform development of targeted therapeutics to attenuate inflammation.

Lack of selective resistance of influenza A virus in presence of host-targeted antiviral, UV-4B.

Development of antiviral drug resistance is a continuous concern for viruses with high mutation rates such as influenza. The use of antiviral drugs targeting host proteins required for viral replication is less likely to result in the selection of resistant viruses than treating with direct-acting antivirals. The iminosugar UV-4B is a host-targeted glucomimetic that inhibits endoplasmic reticulum α-glucosidase I and II enzymes resulting in improper glycosylation and misfolding of viral glycoproteins. UV-4B has broad-spectrum antiviral activity against diverse viruses including dengue and influenza. To examine the ability of influenza virus to develop resistance against UV-4B, mouse-adapted influenza virus was passaged in mice in the presence or absence of UV-4B and virus isolated from lungs was used to infect the next cohort of mice, for five successive passages. Deep sequencing was performed to identify changes in the viral genome during passaging in the presence or absence of UV-4B. Relatively few minor variants were identified within each virus and the ratio of nonsynonymous to synonymous (dN/dS) substitutions of minor variants confirmed no apparent positive selection following sustained exposure to UV-4B. Three substitutions (one synonymous in PB2, one nonsynonymous in M and PA each) were specifically enriched (>3%) in UV-4B-treated groups at passage five. Recombinant viruses containing each individual or combinations of these nonsynonymous mutations remained sensitive to UV-4B treatment in mice. Overall, these data provide evidence that there is a high genetic barrier to the generation and selection of escape mutants following exposure to host-targeted iminosugar antivirals.