Pamela Österlund

IFN Regulatory Factor Family Members Differentially Regulate the Expression of Type III IFN (IFN-λ) Genes

Virus replication induces the expression of antiviral type I (IFN-αβ) and type III (IFN-λ1–3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-λ1 and IFN-λ3 gene promoters revealed them to be similar to IFN-β and IFN-α genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-κB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-λ1 promoter, whereas the IFN-λ3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-λ1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-β gene, whereas IFN-λ2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-α genes.

Gene Expression and Antiviral Activity of Alpha/Beta Interferons and Interleukin-29 in Virus-Infected Human Myeloid Dendritic Cells

ABSTRACT Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-α), alpha/beta interferon (IFN-α/β), and IFN-like interleukin-28A/B (IFN-λ2/3) and IL-29 (IFN-λ1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-α, IFN-α/β, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-α, IFN-α, IFN-β, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-α. IFN-α priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-κB to the respective NF-κB elements of the promoters of IFN-β and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-α or IFN-β. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.

Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons

ABSTRACT In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.

Multiple signaling pathways contribute to synergistic TLR ligand‐dependent cytokine gene expression in human monocyte‐derived macrophages and dendritic cells

TLRs are innate immune receptors that recognize pathogen‐associated structures. Binding of ligands to different TLRs can induce the production of proinflammatory cytokines in a synergistic manner. We have analyzed the molecular mechanisms of synergy in TLR ligand‐stimulated human monocyte‐derived macrophages and dendritic cells (moDCs). Stimulation of moDCs with the TLR8 ligand together with the TLR3 or TLR4 ligand led to synergistic IL‐6, IL‐10, IL‐12, and TNF‐α mRNA expression and cytokine production. DNA‐binding assays showed that TLR3 and TLR8 stimulation induced binding of multiple IFN regulatory factor (IRF) and STAT transcription factors to the IL‐12p35 gene promoter IFN‐stimulated response element in moDCs and macrophages but with different binding profiles and kinetics. We also demonstrate that NF‐κB, MAPKs and PI‐3K pathways have an important role in TLR‐induced cytokine gene expression, as pharmacological inhibitors of these signaling pathways inhibited TLR3, TLR4, and TLR8 ligand‐induced cytokine mRNA expression and protein production. Especially, synergistic IL‐12p70 production was abolished completely in NF‐κB, MAPK p38, and PI‐3K inhibitor‐treated moDCs. Our data suggest that TLR‐dependent, synergistic cytokine gene expression results from enhanced activation and cooperation among NF‐κB, IRF, MAPK, PI‐3K, and STAT signaling pathways.

Cellular Immunity to Mumps Virus in Young Adults 21 Years after Measles-Mumps-Rubella Vaccination

BACKGROUND Measles-mumps-rubella (MMR) vaccination has decreased the incidence of measles, mumps, and rubella virus infections in several countries. However, the persistence of MMR vaccine-induced immunity in the absence of endemic infection has remained unknown. METHODS The persistence of cellular and humoral immunity to mumps virus was studied in 50 individuals (group A) who had been vaccinated twice with MMR vaccine during early childhood and were followed up for 21 years after their first vaccination. Eleven individuals (group B) with naturally acquired immunity to mumps virus were studied for comparison. RESULTS Anti-mumps virus IgG antibodies were detectable (titer > or = 230) in 72% of the vaccinees. A mumps antigen-specific lymphoproliferative response (defined as a stimulatory index [SI] > or = 3) was observed in 98% of group A subjects (mean+/-SD SI, 26+/-30 [range, 0.5-252]) and in 100% of group B subjects (mean+/-SD SI, 22+/-27 [range, 5-123]). Significant mumps antigen-specific interferon- gamma production was detected in 73% of subjects in both groups A and B, and interleukin-10 production was detected in 40% and 36% of group A and B subjects, respectively. CONCLUSIONS All presently seronegative vaccinees (n=14) had mumps antigen-specific lymphoproliferative responses, and only 1 of the seropositive vaccinees (n=36) was devoid of detectable cellular immunity. The results suggest a very long persistence of vaccine-induced anti-mumps virus cellular immunity.

Severe Acute Respiratory Syndrome Coronavirus Fails To Activate Cytokine-Mediated Innate Immune Responses in Cultured Human Monocyte-Derived Dendritic Cells

ABSTRACT Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-α], IFN-β, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-α/β-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.

TLR ligands induce synergistic interferon-β and interferon-λ1 gene expression in human monocyte-derived dendritic cells

Toll-like receptors (TLRs) are pattern-recognition receptors of the innate immune system that recognize various pathogen-associated molecules. TLR ligands are potent activators of immune cells and certain TLR ligands have a synergistic ability to induce the production of pro-inflammatory cytokines. In the present study we have analyzed the potential synergy between TLR3, TLR4 and TLR7/8 ligands in type I and type III interferon (IFN) gene expression in human monocyte-derived dendritic cells (moDCs). We show that stimulation of moDCs with TLR7/8 ligand R848 together with TLR3 or TLR4 ligands, polyI:C or LPS, respectively, leads to a synergistic expression of IFN-β and IFN-λ1 mRNAs. Neutralization of type I IFNs as well as IFN priming prior to stimulation suggest that IFN-dependent positive feedback loop is at least partly responsible for the mechanism of synergy. Enhanced expression of TLR3 and especially TLR7, which are both under the regulation of type I IFNs, correlated to synergistic TLR ligand-dependent induction of IFN-β and IFN-λ1 genes. NF-κB, PI3 kinase and MAP kinase pathways were involved in TLR ligand-induced IFN gene expression as evidenced by pharmacological signaling inhibitors. The data indicates that IFNs contribute to TLR-dependent gene activation in human DCs stimulated with multiple TLR ligands.

Innate Immune Responses in Human Monocyte-Derived Dendritic Cells Are Highly Dependent on the Size and the 5′ Phosphorylation of RNA Molecules

Recognition of viral genetic material takes place via several different receptor systems, such as retinoic acid-inducible gene I-like receptors and TLRs 3, 7, 8, and 9. At present, systematic comparison of the ability of different types of RNAs to induce innate immune responses in human immune cells has been limited. In this study, we generated bacteriophage ϕ6 and influenza A virus-specific ssRNA and dsRNA molecules ranging from 58 to 2956 nt. In human monocyte-derived dendritic cells (moDCs), short dsRNAs efficiently upregulated the expression of IFN (IFN-α, IFN-β, and IFN–λ1) and proinflammatory (TNF-α, IL-6, IL-12, and CXCL10) cytokine genes. These genes were also induced by ssRNA molecules, but size-specific differences were not as pronounced as with dsRNA molecules. Dephosphorylation of short ssRNA and dsRNA molecules led to a dramatic reduction in their ability to stimulate innate immune responses. Such a difference was not detected for long ssRNAs. RNA-induced cytokine responses correlated well with IFN regulatory factor 3 phosphorylation, suggesting that IFN regulatory factor 3 plays a major role in both ssRNA- and dsRNA-activated responses in human moDCs. We also found that IFN gene expression was efficiently stimulated following recognition of short dsRNAs by retinoic acid-inducible gene I and TLR3 in human embryonic kidney 293 cells, whereas ssRNA-induced responses were less dependent on the size of the RNA molecule. Our data suggest that human moDCs are extremely sensitive in recognizing foreign RNA, and the responses depend on RNA size, form (ssRNA versus dsRNA), and the level of 5′ phosphorylation.

Cytokine responses in cord blood predict the severity of later respiratory syncytial virus infection.

BACKGROUND It has been claimed that an early respiratory syncytial virus (RSV) infection can induce asthma and recurrent wheezing. OBJECTIVE We addressed the question of whether infants contracting an early RSV infection differ from healthy children in their cytokine production at birth. METHODS In a prospective cohort study cord blood samples were collected from 1084 newborns during autumn 2001. Of 47 of these newborns with subsequent virologically confirmed RSV infection before 6 months of age, 24 had enough cells for stimulation in cord blood samples (14 of those were hospitalized). Twenty-eight children had other respiratory virus infections (16 with enough cells), and samples from 48 healthy children of the 1084 total served as control specimens. Stimulated cytokine production of mononuclear cells was measured. The responses in the groups were evaluated by means of factor analysis. RESULTS The infants hospitalized for RSV infection had higher LPS-stimulated combined IL-6 and IL-8 responses than the infants treated as outpatients (P = .005) or the healthy control subjects (P = .02). The hospitalized patients with RSV showed lower IL-1beta, IL-2, IL-4, IL-5, and IL-10 responses than those treated as outpatients (P = .02). High IL-6 and IL-8 responsiveness predicted a severe RSV infection (odds ratio, 2.20; 95% CI, 1.17-4.14; P = .01). The unstimulated cytokine responses at birth did not differ between the patients and healthy control subjects. CONCLUSION The results suggest that natural differences in innate immunity predispose children to severe RSV infection rather than the infection modifying immune responses in childhood.

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.