Ongoing research focuses on the exploration of pathologic inflammatory responses to acute respiratory virus infection and the use of this information to develop creative strategies to circumvent the lethal sequelae characteristic of this disease. In our first original manuscript of this year, we described the development of a fully functional recombinant PVM strain with a fluorescent reporter protein (rK2-PVM) that permits us to track infection of target cells in vivo. With rK2-PVM, we demonstrate infection of leukocytes in the lung, notably, dendritic cells and alveolar macrophages. Alveolar macrophages undergo productive infection and release infectious virions. We have shown previously that administration of immunobiotic Lactobacillus directly to the respiratory mucosa protects mice from the lethal sequelae of PVM infection in association with profound suppression of the virus-induced inflammatory response. We show here that Lactobacillus administration also limits infection of leukocytes in vivo and results in diminished release of infectious virions from alveolar macrophages. This is the first study to provide insight into the cellular basis of the antiviral impact of immunobiotic L. plantarum. (Dyer et al. 2015 J. Virol.90(2):979-91) In our second manuscript, we continued our examination of the interactions of Lactobacillus strains with alveolar macrophages (AMs), specifically with the MH-S model AM cell line. Specifically, we found that the SV-40-transformed MH-S cell line maintains some, but not all, features of primary alveolar macrophages (AMs) from BALB/c mice. We found that MH-S cells produce inflammatory cytokines IL-6 and CXCL10 in response to challenge with Gram-positive Lactobacillus reuteri, and to TLR2 and NOD2 ligands Pam3CSK4 and MDP, respectively. In contrast, although wild-type AMs are infected in vivo by pneumonia virus of mice (PVM), no virus replication was detected in MH-S cells. Interestingly, the surface immunophenotype of MH-S cells (CD11c(+)Siglec F(-)) differs from that of wild-type AMs (CD11c(+) Siglec F(+)) and is similar to that of immature AMs isolated from granulocyte macrophage-colony stimulating factor (GM-CSF) gene-deleted mice; AMs from GM-CSF(-/-) mice also support PVM replication. However, MH-S cells do not express the GM-CSF receptor alpha chain (CD116) and do not respond to GM-CSF. Due to these unusual features, MH-S cells should be used with caution as experimental models of AMs. (Brenner et al. 2016. Immunol. Lett. 172:106-12) In our third original manuscript of this cycle, we continued our exploration of the interactions of Lactobacillus with the host in vivo. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2-/- and NOD2-/- mice. In this manuscript we demonstrated substantial loss of Lp-mediated protection in a double gene-deleted NOD2-/-TLR2-/- strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2-/-TLR2-/- mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory infection.
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