Sinikka Latvala

Live Lactobacillus rhamnosus and Streptococcus pyogenes differentially regulate Toll‐like receptor (TLR) gene expression in human primary macrophages

Macrophages are phagocytes that recognize bacteria and subsequently activate appropriate innate and adaptive immune responses. TLRs are essential in identifying conserved bacterial structures and in initiating and mediating innate immune responses. In this work, we have characterized TLR gene expression in human monocyte‐derived macrophages in response to stimulation with two live Gram‐positive bacteria, a human commensal and probiotic Lactobacillus rhamnosus GG (LGG), and an important human pathogen Streptococcus pyogenes. LGG and S. pyogenes enhanced TLR2 expression in macrophages. LGG and S. pyogenes also required TLR2 for NF‐κB activation. Only pathogenic S. pyogenes was able to up‐regulate TLR3 and TLR7 gene expression. This up‐regulation was dependent on IFN‐α/β, as neutralizing anti‐IFN‐α/β antibodies reduced S. pyogenes‐induced TLR3 and TLR7 mRNA expression. Our results show that despite similarities, TLR responses of macrophages differ for a Gram‐positive probiotic and a pathogen. Our data suggest that macrophages can discriminate between probiotic and pathogenic bacteria by IFN‐mediated TLR gene regulation.

Lactobacillus rhamnosus GG and Streptococcus thermophilus induce suppressor of cytokine signalling 3 (SOCS3) gene expression directly and indirectly via interleukin-10 in human primary macrophages

In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)‐10]/Th1 (IL‐12) cytokine expression balance in human primary macrophages stimulated with multiple non‐pathogenic Gram‐positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti‐inflammatory IL‐10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL‐12 production, while the production of IL‐12 induced by other bacteria remained at a low level. The highest anti‐inflammatory potential was seen with bifidobacteria, as evidenced by high IL‐10/IL‐12 induction ratios. All studied non‐pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria‐induced IL‐10 production, as demonstrated by experiments with cycloheximide (CHX) and anti‐IL‐10 antibodies, respectively. The mitogen‐activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria‐induced SOCS3 gene expression. Enhanced IL‐10 production and SOCS3 gene expression induced by live non‐pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.

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

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.

Inhibition of dynamin-dependent endocytosis interferes with type III IFN expression in bacteria-infected human monocyte-derived DCs

Type I IFNs (IFN‐α/βs) and type III IFNs (IFN‐λ1–3) play an important role in host defense against viral infections. The induction of type I IFNs has recently been found to take place also in bacterial infections, and therefore, this study focuses on analyzing the regulation of type III IFNs in response to bacterial stimulation. We found by quantitative RT‐PCR that the expression of IFN‐λ1 and IFN‐λ2/3 mRNAs, as well as that of IFN‐β, was similarly up‐regulated in response to stimulation with live Salmonella typhimurium or TLR4 agonist LPS in human moDCs. The induction of IFN‐λ mRNAs did not require ongoing protein synthesis, and only IFN‐λ1 was detected at the protein level. The induction of IFN‐λ mRNAs was sensitive to SB202190, Ly294002, and PDTC, which inhibit p38 MAPK, PI3K, and NF‐κB activation, respectively. Furthermore, we observed that blocking dynamin‐dependent endocytosis pathways with dynasore led to decreased cell surface expression of CD86 and HLA class II molecules and reduced production of IFN‐λ1, CXCL10, and IL‐6 when the cells were infected with S. typhimurium. Cytokine production was also impaired in dynasore‐treated, Streptococcus thermophilus‐stimulated cells. Further, inhibition of dynamin prevented S. typhimurium‐induced phosphorylation of IRF3 and the internalization of the bacteria. In summary, induction of type III IFNs in bacteria‐infected human moDCs requires multiple signaling pathways and involves bacterial phagocytosis.

RIG-I Signaling Is Essential for Influenza B Virus-Induced Rapid Interferon Gene Expression.

ABSTRACT Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a comparison of influenza A and B virus infection mechanisms may provide new insight into virus-host interactions. Here we analyzed the early events in influenza B virus infection and interferon (IFN) gene expression in human monocyte-derived macrophages and dendritic cells. We show that influenza B virus induces IFN regulatory factor 3 (IRF3) activation and IFN-λ1 gene expression with faster kinetics than does influenza A virus, without a requirement for viral protein synthesis or replication. Influenza B virus-induced activation of IRF3 required the fusion of viral and endosomal membranes, and nuclear accumulation of IRF3 and viral NP occurred concurrently. In comparison, immediate early IRF3 activation was not observed in influenza A virus-infected macrophages. Experiments with RIG-I-, MDA5-, and RIG-I/MDA5-deficient mouse fibroblasts showed that RIG-I is the critical pattern recognition receptor needed for the influenza B virus-induced activation of IRF3. Our results show that innate immune mechanisms are activated immediately after influenza B virus entry through the endocytic pathway, whereas influenza A virus avoids early IRF3 activation and IFN gene induction. IMPORTANCE Recently, a great deal of interest has been paid to identifying the ligands for RIG-I under conditions of natural infection, as many previous studies have been based on transfection of cells with different types of viral or synthetic RNA structures. We shed light on this question by analyzing the earliest step in innate immune recognition of influenza B virus by human macrophages. We show that influenza B virus induces IRF3 activation, leading to IFN gene expression after viral RNPs (vRNPs) are released into the cytosol and are recognized by RIG-I receptor, meaning that the incoming influenza B virus is already able to activate IFN gene expression. In contrast, influenza A (H3N2) virus failed to activate IRF3 at very early times of infection, suggesting that there are differences in innate immune recognition between influenza A and B viruses.

Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages

In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT‐domain‐, leucine‐rich repeat‐ and PYD‐containing protein 3 (NALP3) inflammasome complex in human monocyte‐derived macrophages and the molecules and signalling pathways involved in GAS‐induced inflammatory responses. We focused upon analysing the impact of dynamin‐dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome–lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild‐type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)‐1β, IL‐10, tumour necrosis factor (TNF)‐α and chemokine (C‐X‐C motif) ligand (CXCL)‐10 cytokines were produced after bacterial stimulation in a dose‐dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin‐dependent endocytosis in macrophages attenuates the induction of IL‐1β, TNF‐α, interferon (IFN)‐β and CXCL‐10 mRNAs. We also observed that pro‐IL‐1β protein was expressed and efficiently cleaved into mature‐IL‐1β via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS‐stimulated inflammatory responses in human macrophages.