Priming of the Respiratory Tract with ImmunobioticLactobacillus plantarumLimits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM)

Abstract

Pneumonia virus of mice (PVM) is a natural rodent pathogen that replicates in bronchial epithelial cells and reproduces manyclinical and pathological features of the more severe forms of disease associated with human respiratory syncytial virus. In order to track virus-target cell interactions during acute infection in vivo, we developed rK2-PVM, bacterial artificial chromosomebased recombinant PVM strain J3666 that incorporates the fluorescent tag monomeric Katushka 2 (mKATE2). The rK2-PVM pathogen promotes lethal infection in BALB/c mice and elicits characteristic cytokine production and leukocyte recruitment to the lung parenchyma. Using recombinant virus, we demonstrate for the first time PVM infection of both dendritic cells (DCs; CD11c ؉ major histocompatibility complex class II ؉ ) and alveolar macrophages (AMs; CD11c ؉ sialic acid-binding immunoglobulin-like lectin F ؉ ) in vivo and likewise detect mKATE2 ؉ DCs in mediastinal lymph nodes from infected mice. AMs support both active virus replication and production of infectious virions. Furthermore, we report that priming of the respiratory tract with immunobiotic Lactobacillus plantarum, a regimen that results in protection against the lethal inflammatory sequelae of acute respiratory virus infection, resulted in differential recruitment of neutrophils, DCs, and lymphocytes to the lungs in response to rK2-PVM and a reduction from ϳ40% to <10% mKATE2 ؉ AMs in association with a 2-log drop in the release of infectious virions. In contrast, AMs from L. plantarum-primed mice challenged with virus ex vivo exhibited no differential susceptibility to rK2-PVM. Although the mechanisms underlying Lactobacillus-mediated viral suppression remain to be fully elucidated, this study provides insight into the cellular basis of this response. H uman respiratory syncytial virus (hRSV, family Paramyxoviridae, genus Pneumovirus) is a major cause of morbidity and death among infants and children worldwide and is recognized as a significant health concern among the elderly (1). The anti-hRSV antibody paluvizumab is highly effective when used as prophylaxis in infants identified as high risk (2), although there are many infants hospitalized with severe hRSV disease who are not identified in any of the high-risk cohorts (3). Treatment options for hRSV disease are primarily supportive. The antiviral agent ribavirin is currently recommended only for severely ill and immunocompromised children (4, 5). New antiviral agents that focus specifically on hRSV are in development (6)(7)(8), and the inflammatory responses characteristic of severe hRSV disease are also recognized as targets for therapeutic intervention (9, 10).Pneumonia virus of mice (PVM) is a rodent pathogen of the same family and genus as hRSV. PVM infection in inbred strains of mice reproduces many of the clinical and pathological features of the more severe forms of hRSV disease, which has facilitated the exploration of new therapeutic strategies in vivo (11,12). Similar to hRSV (13), PVM infects bronchial epi...

Immortalized MH-S cells lack defining features of primary alveolar macrophages and do not support mouse pneumovirus replication

“…PVM undergoes robust replication in the lung tissue of inbred strains of mice [16 – 18], as well as productive replication in alveolar macrophages in vivo [19]. PVM also replicates in the mouse peritoneal macrophage RAW 264.7 cell line [45, 19], although no replication was detected in MH-S cells challenged with PVM under similar conditions [Fig.…”

“…Recombinant rK2-PVM (strain J3666 with mKATE2 fluorescent tag) was generated as described [19]. Lactobacillus reuteri F275 (ATCC 23272) was grown overnight in Mann Rogosa Sharpe broth at 37°C; the ratio of OD 600 to colony forming units was determined experimentally [20].…”

“…Stationary phase cultures were heat-inactivated at 70°C for 30 min, washed with PBS/BSA, and stored at −80°C at 5 × 10 9 cells / mL prior to use. Initial titration of (recombinant) rK2-PVM in tissue culture was performed by a modification of the TCID 50 assay in which virus infection is monitored by detection of the mKATE2 fluorescent marker (TCFD 50 ; see reference 19). …”

“…PVM infection is lethal in numerous inbred strains of mice [16], with inflammatory pathology characterized by robust viral replication in the bronchiolar epithelium, granulocyte recruitment to the airways, and local production of proinflammatory mediators, including CCL2, CCL3 and CXCL1 [17, 18]. Using recombinant PVM (rK2-PVM) which incorporates the sequence encoding the far-red fluorescent protein, mKATE2, we have recently shown that AMs are a primary leukocyte target for this virus, and that AMs support both virus replication and productive infection in vivo [19]. Furthermore, application of immunobiotic Lactobacillus species to the respiratory tract, a regimen that results in robust and sustained survival in response to virus infection [20 – 23] limits PVM infection and virion release from AMs [19].…”

“…Using recombinant PVM (rK2-PVM) which incorporates the sequence encoding the far-red fluorescent protein, mKATE2, we have recently shown that AMs are a primary leukocyte target for this virus, and that AMs support both virus replication and productive infection in vivo [19]. Furthermore, application of immunobiotic Lactobacillus species to the respiratory tract, a regimen that results in robust and sustained survival in response to virus infection [20 – 23] limits PVM infection and virion release from AMs [19]. …”