Human milk oligosaccharides (HMO) contribute to innate immunity by enhancing growth of beneficial bacteria, epithelial cell maturation and mucosal barrier integrity. They have immunomodulatory effects and can block pathogen binding to host cell surface glycans or receptors. We investigated the effects of 2'-fucosyllactose (2'FL), 6'-sialyllactose (6'SL), 3'-sialyllactose (3'SL) and lacto-N-neoTetraose (LNnT) on human respiratory epithelial cell lines or peripheral blood mononuclear cells (PBMCs) following respiratory viral infection in vitro. Expression of cytokines and viral load were monitored in infected cells. These biomarkers of innate immunity were selected since viral load and cytokine levels (IP-10, MIP-1α, IL-6, IL-8, TNF-α) have been correlated with disease severity in respiratory syncytial virus (RSV) and influenza (IAV) virus infection in vivo. 2'FL significantly decreased RSV viral load and cytokines associated with disease severity (IL-6, IL-8, MIP-1α) and inflammation (TNF-α, MCP-1) in airway epithelial cells. LNnT and 6'SL significantly decreased IAV viral load in airway epithelial cells. 6'SL dose-dependently down-regulated IP-10 and TNF-α in RSV infected PBMCs. HMO at or below levels found in breast milk enhance innate immunity to respiratory viruses in vitro and may interact directly with cells to modulate biomarkers of innate immunity.

BackgroundIndividuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV.MethodsIn order to determine requirements for correction of RSV induced injury we have generated compound transgenic mice where SP-C expression can be induced on the Sftpc -/- background (SP-C/Sftpc -/-) by the administration of doxycycline (dox). The pattern of induced SP-C expression was determined by immunohistochemistry and processing by Western blot analysis. Tissue and cellular inflammation was measured following RSV infection and the RSV-induced cytokine response of isolated Sftpc +/+ and -/- type II cells determined.ResultsAfter 5 days of dox administration transgene SP-C mRNA expression was detected by RT-PCR in the lungs of two independent lines of bitransgenic SP-C/Sftpc -/- mice (lines 55.3 and 54.2). ProSP-C was expressed in the lung, and mature SP-C was detected by Western blot analysis of the lavage fluid from both lines of SP-C/Sftpc -/- mice. Induced SP-C expression was localized to alveolar type II cells by immunostaining with an antibody to proSP-C. Line 55.3 SP-C/Sftpc -/- mice were maintained on or off dox for 7 days and infected with 2.6x107 RSV pfu. On day 3 post RSV infection total inflammatory cell counts were reduced in the lavage of dox treated 55.3 SP-C/Sftpc -/- mice (p = 0.004). The percentage of neutrophils was reduced (p = 0.05). The viral titers of lung homogenates from dox treated 55.3 SP-C/Sftpc -/- mice were decreased relative to 55.3 SP-C/Sftpc -/- mice without dox (p = 0.01). The cytokine response of Sftpc -/- type II cells to RSV was increased over that of Sftpc +/+ cells.ConclusionsTransgenic restoration of SP-C reduced inflammation and improved viral clearance in the lungs of SP-C deficient mice. The loss of SP-C in alveolar type II cells compromises their response to infection. These findings show that the restoration of SP-C in Sftpc -/- mice in response to RSV infection is a useful model to determine parameters for therapeutic intervention.

Pulmonary surfactant protein-C (SP-C) gene-targeted mice (Sftpc 2/2 ) develop progressive lung inflammation and remodeling. We hypothesized that SP-C deficiency reduces the ability to suppress repetitive inflammatory injury. Sftpc 1/1 and Sftpc 2/2 mice given three doses of bacterial LPS developed airway and airspace inflammation, which was more intense in the Sftpc 2/2 mice at 3 and 5 days after the final dose. Compared with Sftpc 1/1 mice, inflammatory injury persisted in the lungs of Sftpc 2/2 mice 30 days after the final LPS challenge. Sftpc 2/2 mice showed LPS-induced airway goblet cell hyperplasia with increased detection of Sam pointed Ets domain and FoxA3 transcription factors. Sftpc 2/2 type II alveolar epithelial cells had increased cytokine expression after LPS exposure relative to Sftpc 1/1 cells, indicating that type II cell dysfunction contributes to inflammatory sensitivity. Microarray analyses of isolated type II cells identified a pattern of enhanced expression of inflammatory genes consistent with an intrinsic low-level inflammation resulting from SP-C deficiency. SP-C-containing clinical surfactant extract (Survanta) or SP-C/phospholipid vesicles blocked LPS signaling through the LPS receptor (Toll-like receptor [TLR] 4/CD14/MD2) in human embryonic kidney 293T cells, indicating that SP-C blocks LPS-induced cytokine production by a TLR4-dependent mechanism. Phospholipid vesicles alone did not modify the TLR4 response. In vivo deficiency of SP-C leads to inflammation, increased cytokine production by type II cells, and persistent inflammation after repetitive LPS stimulation.Keywords: surfactant protein-C; LPS; lung inflammation; type II cells; Toll-like receptor 4Surfactant protein-C (SP-C) is an abundant 3.5-kD surfactantassociated protein expressed and secreted by alveolar type II epithelial cells. The mature airspace form of SP-C is highly hydrophobic and palmitoylated at adjacent amino terminal cysteine residues (1). Mutations in the gene encoding human SP-C (SFTPC) have been shown to cause familial interstitial lung disease (ILD). Affected individuals express an altered proSP-C, leading to reduced levels of mature SP-C in the airspace (2-4). SP-C deficiencies without detectable mutations in the protein-coding region of SFTPC or due to promoter mutations have also been reported, and represent a true null condition (5-7). These individuals also develop ILD, including idiopathic pulmonary fibrosis (IPF). SP-C deficiency-related disease may arise as an acute childhood or as a late-onset disease in adulthood (3). Because SP-C is expressed exclusively in alveolar type II cells, the associated ILD/idiopathic pulmonary fibrosis presumably originates from a primary defect in the epithelium. SP-C deficiency increases susceptibility to viral-or bacterial-induced exacerbations in affected children (5,6,(8)(9)(10). SP-C-deficient mice (Sftpc 2/2 ) developed a strain-specific pulmonary phenotype with age that was similar to the human disease (11). Sftpc 2/2 mice were more susceptible to challenge with ...

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