Mari Strengell

IL-21 in Synergy with IL-15 or IL-18 Enhances IFN-γ Production in Human NK and T Cells

NK and T cell-derived IFN-γ is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-γ, T-bet, IL-12Rβ2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-γ gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-γ mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-γ gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-γ gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-γ production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-γ gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-γ gene regulatory sites. IL-18, however, activated the binding of NF-κB to the IFN-γ promoter NF-κB site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.

IL-21 Up-Regulates the Expression of Genes Associated with Innate Immunity and Th1 Response

IL-21 is a recently characterized T cell-derived cytokine that regulates NK and T cell function. IL-21R shares the common γ-chain (γc) with the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Despite the same γc, these cytokines have different effects on diverse cells. In this study, we have studied IL-15- and IL-21-induced gene expression in human primary NK and T cells and the NK-92 cell line. Both IL-15 and IL-21 rapidly induced mRNA synthesis for IFN-γ, T-bet, IL-2Rα, IL-12Rβ2, IL-18R, and myeloid differentiation factor 88 (MyD88), the genes that are important in activating innate immunity and Th1 response. IL-15 induced STAT5 DNA binding to the IL-2Rα IFN-γ-activated sequence (GAS), MyD88 GAS, and c-sis-inducible elements, whereas IL-21 induced STAT3 DNA binding to MyD88 GAS and c-sis-inducible elements. IL-21-induced STAT3 activation was verified by immunoprecipitation and Western blotting with anti-phosphotyrosine Ab. In addition, pretreatment of NK-92 cells with IL-15 or IL-21 strongly enhanced IL-12-induced STAT4 DNA binding to IL-2Rα GAS. The induction of IFN-γ, T-bet, IL-12Rβ2, and IL-18R gene expression in NK cells, along with STAT3 activation, suggests that IL-21 is involved in the activation of innate immune responses. Moreover, the enhanced transcription of these genes in T cells establishes a significant role for IL-21 also in the Th1 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.

IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells

Dendritic cells (DCs) play an important role in innate and adaptive immune responses. In addition to their phagocytic activity, DCs present foreign antigens to naïve T cells and regulate the development of adaptive immune responses. Upon contact with DCs, activated T cells produce large quantities of cytokines such as interferon‐γ (IFN‐γ) and interleukin (IL)‐21, which have important immunoregulatory functions. Here, we have analyzed the effect of IL‐21 and IFN‐γ on lipopolysaccharide (LPS)‐induced maturation and cytokine production of human monocyte‐derived DCs. IL‐21 and IFN‐γ receptor genes were expressed in high levels in immature DCs. Pretreatment of immature DCs with IL‐21 inhibited LPS‐stimulated DC maturation and expression of CD86 and human leukocyte antigen class II (HLAII). IL‐21 pretreatment also dramatically reduced LPS‐stimulated production of tumor necrosis factor α, IL‐12, CC chemokine ligand 5 (CCL5), and CXC chemokine ligand 10 (CXCL10) but not that of CXCL8. In contrast, IFN‐γ had a positive feedback effect on immature DCs, and it enhanced LPS‐induced DC maturation and the production of cytokines. IL‐21 weakly induced the expression Toll‐like receptor 4 (TLR4) and translation initiation region (TIR) domain‐containing adaptor protein (TIRAP) genes, whereas the expression of TIR domain‐containing adaptor‐inducing IFN‐β (TRIF), myeloid differentiation (MyD88) 88 factor, or TRIF‐related adaptor molecule (TRAM) genes remained unchanged. However, IL‐21 strongly stimulated the expression of suppressor of cytokine signaling (SOCS)‐1 and SOCS‐3 genes. SOCS are known to suppress DC functions and interfere with TLR4 signaling. Our results demonstrate that IL‐21, a cytokine produced by activated T cells, can directly inhibit the activation and cytokine production of myeloid DCs, providing a negative feedback loop between DCs and T lymphocytes.

Minor Changes in the Hemagglutinin of Influenza A(H1N1)2009 Virus Alter Its Antigenic Properties

Background The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination. Methods/Results Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20th century. Conclusions According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.

IFN-α regulates IL-21 and IL-21R expression in human NK and T cells

Interleukin (IL)‐21 is a T cell‐derived cytokine that regulates innate and adaptive immune responses. IL‐21 receptor (IL‐21R), which is expressed in natural killer (NK) and T cells, is structurally homologous to IL‐2Rβ and IL‐15Rα. These receptors also share a common cytokine receptor γ‐chain with IL‐4, IL‐7, and IL‐9. Macrophage‐ or dendritic cell‐derived interferon (IFN)‐α/β is a key cytokine in regulation of NK and T cell functions. We demonstrate here that in addition to activating IFN‐γ gene expression, IFN‐α/β and IL‐12 enhance the mRNA expression of IL‐21 in activated human T cells. In addition, IFN‐α/β enhanced T cell receptor stimulation‐induced IL‐21 and IFN‐γ gene expression in resting T cells. The promoter analysis of IL‐21 gene revealed a putative IFN‐γ activation site element, which was found to bind signal transducer and activator of transcription 1 (STAT1), STAT2, STAT3, and STAT4 proteins in IFN‐α/β‐stimulated NK or T cell extracts. In contrast to IL‐21 expression, IFN‐α/β down‐regulated IL‐21R mRNA expression in NK and T cells. IFN‐α/β‐induced down‐regulation of IL‐21R expression resulted in reduced STAT3 phosphorylation and DNA binding after IL‐21 stimulation. In conclusion, our results suggest a novel role for IFN‐α/β in the regulation of IL‐21 response.

Critical immunological pathways are downregulated in APECED patient dendritic cells

Autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is a monogenic autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene. AIRE functions as a transcriptional regulator, and it has a central role in the development of immunological tolerance. AIRE regulates the expression of ectopic antigens in epithelial cells of the thymic medulla and has been shown to participate in the development of peripheral tolerance. However, the mechanism of action of AIRE has remained elusive. To further investigate the role of AIRE in host immune functions, we studied the properties and transcript profiles in in vitro monocyte-differentiated dendritic cells (moDCs) obtained from APECED patients and healthy controls. AIRE-deficient monocytes showed typical DC morphology and expressed DC marker proteins cluster of differentiation 86 and human leukocyte antigen class II. APECED patient-derived moDCs were functionally impaired: the transcriptional response of cytokine genes to pathogens was drastically reduced. Interestingly, some changes were observable already at the immature DC stage. Pathway analyses of transcript profiles revealed that the expression of the components of the host cell signaling pathways involved in cell–cell signalling, innate immune responses, and cytokine activity were reduced in APECED moDCs. Our observations support a role for AIRE in peripheral tolerance and are the first ones to show that AIRE has a critical role in DC responses to microbial stimuli in humans.

Antibody responses against influenza A(H1N1)pdm09 virus after sequential vaccination with pandemic and seasonal influenza vaccines in Finnish healthcare professionals

Background  Influenza A(H1N1)pdm09 virus has been circulating in human population for three epidemic seasons. During this time, monovalent pandemic and trivalent seasonal influenza vaccination against this virus have been offered to Finnish healthcare professionals. It is, however, unclear how well vaccine‐induced antibodies recognize different strains of influenza A(H1N1)pdm09 circulating in the population and whether the booster vaccination with seasonal influenza vaccine would broaden the antibody cross‐reactivity.

Incoming Influenza A Virus Evades the Early Host Recognition – Direct Interferon Induction by Influenza B Virus Entry

ABSTRACT The activation of the interferon (IFN) system, which is triggered largely by the recognition of viral nucleic acids, is one of the most important host defense reactions against viral infections. Although influenza A and B viruses, which both have segmented negative-strand RNA genomes, share major structural similarities, they have evolutionarily diverged, with total genetic incompatibility. Here we compare antiviral-inducing mechanisms during infections with type A and B influenza viruses in human dendritic cells. We observed that IFN responses are induced significantly faster in cells infected with influenza B virus than in cells infected with type A influenza virus and that the early induction of antiviral gene expression is mediated by the activation of the transcription factor IFN regulatory factor 3 (IRF3). We further demonstrate that influenza A virus infection activates IFN responses only after viral RNA (vRNA) synthesis, whereas influenza B virus induces IFN responses even if its infectivity is destroyed by UV treatment. Thus, initial viral transcription, replication, and viral protein synthesis are dispensable for influenza B virus-induced antiviral responses. Moreover, vRNA molecules from both type A and B viruses are equally potent activators of IFN induction, but incoming influenza B virus structures are recognized directly in the cytosol, while influenza A virus is able to evade early recognition. Collectively, our data provide new evidence of a novel antiviral evasion strategy for influenza A virus without a contribution of the viral NS1 protein, and this opens up new insights into different influenza virus pathogenicities.