Nonpathogenic Lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages

Abstract

In this study, we have utilized global gene expression profiling to compare the responses of human primary macrophages to two closely related, well-characterized Lactobacillus rhamnosus strains GG and LC705, since our understanding of the responses elicited by nonpathogenic bacteria in human innate immune system is limited. Macrophages are phagocytic cells of the innate immune system that perform sentinel functions to initiate appropriate responses to surrounding stimuli. Macrophages that reside on gut mucosa encounter ingested and intestinal bacteria. Bacteria of Lactobacillus genus are nonpathogenic and used in food and as supplements with health-promoting probiotic potential. Our results demonstrate that live GG and LC705 induced quantitatively different gene expression profiles in macrophages. A gene ontology analysis revealed functional similarities and differences in responses to GG and LC705 that were reflected in host defense responses. Both GG and LC705 induced interleukin-1β production in macrophages that required caspase-1 activity. LC705, but not GG, induced type I interferon -dependent gene activation that correlated with its ability to prevent influenza A virus replication and production of viral proteins in macrophages. Our results indicate that nonpathogenic bacteria are able to activate the inflammasome. In addition, our results suggest that L. rhamnosus may prime the antiviral potential of human macrophages.

Figures

Figure 1. Functional annotation of the biological processes affected in macrophages stimulated by live GG or LC705. Depicted are immunologically relevant GO-classes that are significantly affected by either GG or LC705. P-values below or equal to 0.01 were considered as significant. Dotted lines indicate p-value of 0.01. David software was used in the assessment of categories as described in Materials and Methods. The complete list of GO-classes affected is available in the Supplemental Materials.

Figure 2. Expression profiles of selected genes significantly modulated by GG or LC705 stimulation in macrophages. Heat map shows genes that were significantly differentially expressed (p ≤ 0.01), and were also present in functional GO-classes activated by GG or LC705. Clustering and visualization were obtained with TMEV-tool.

Figure 3. Expression of NALP3 inflammasome components in macrophages is activated by both GG and LC705. (A) Human primary macrophages were stimulated at a 10:1 (bacteria:macrophage) ratio with GG or LC705, and total RNA was isolated for qRT-PCR analysis. Results were normalized to β-actin levels, and relative fold induction was determined by using the 2-ΔΔCT formula. Results are presented as means ± SD from three independent experiments, each performed with cells from six donors (B) Human macrophages were treated as described above, and cell lysates were prepared. Proteins were separated on 15% SDS-PAGE, blotted, and visualized with indicated antibodies. Results are representative of four independent experiments.

Figure 4. Processing and secretion of IL-1β occurs upon stimulation of macrophages by live and dead lactobacilli. (A) Human primary macrophages were stimulated at 2:1, 10:1, and 25:1 (bacteria:macrophages) ratios with GG or LC705. Proteins were separated with 15% SDS-PAGE, blotted, and visualized with indicated antibodies. (B) Production of IL1β was measured from cell culture supernatants by ELISA at 6 and 24 h after stimulation. The results are the means ± SD from a representative experiment performed with cells from six donors.

Figure 5. Caspase-1 activity is required for GG and LC705 induced IL-1β production in macrophages. (A) Human primary macrophages were treated for 30 min with 50 μM Ac-YVAD-CHO caspase-1 inhibitor peptide before stimulation with live GG or LC705 for 6 or 24 h. Supernatants were collected, and the amounts of mature IL1β and TNF-α were determined by ELISA. The results at 6 h are the means ± SD of four donors and the results at 24 h are representative of four independent experiments each performed with cells from four donors. (B) Dynasore was used at 80 μM to pretreat macrophages 30 min prior to 6 h GG or LC705 stimulation. The presence of pro-IL-1β, mature IL-1β, pro-caspase-1 and mature caspase-1 was analyzed by western blotting. (C) Secreted IL-1β was measured from 6 h cell culture supernatants by ELISA. Results are the means ± SD of two independent experiments, each performed with cells from four donors. Calculations for p-values were performed using Anova, and group-wise differences were analyzed by paired t-test followed by FDR-test (*p ≤ 0.1). Only statistically significant differences between groups are marked in the figure.

Figure 6. Antiviral responses are activated by live GG and LC705. (A) Macrophage supernatants were collected at 6 and 24 h after stimulation with GG or LC705, and the amounts of IFN-α and CXCL10 were measured by ELISA. (B) Macrophages were stimulated with live GG and LC705, after which the expression of RIG-I, MxA and MxB was analyzed by western blotting with specific antibodies. The results are representative of four independent experiments, each performed with cells from four blood donors. (C) Macrophages were stimulated with live GG or LC705 for 24 h, followed by influenza A infection for 6 h. Viral replication was measured by using qRT-PCR specific for influenza NP, M1 or NS1 mRNA. The experiment was repeated three times with similar results. (D) Macrophages primed with GG or LC705 for 24 h were infected with indicated doses of influenza A, and cell lysates were collected at 6 h. Influenza A NP, M1 and NS1 proteins were detected by western blotting by using specific antibodies. (E) western blot band intensities from panel D were scanned separately after which NP, M1, and NS1 signal intensities were normalized to the levels of β-actin.