Anne Lehtonen

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.

Lactobacilli and Streptococci Activate NF-κB and STAT Signaling Pathways in Human Macrophages

Gram-positive bacteria induce the production of several cytokines in human leukocytes. The molecular mechanisms involved in Gram-positive bacteria-induced cytokine production have been poorly characterized. In this work we demonstrate that both nonpathogenic Lactobacillus rhamnosus GG and pathogenic Streptococcus pyogenes (group A streptococci) induce NF-κB and STAT DNA-binding activity in human primary macrophages as analyzed by EMSA. NF-κB activation was rapid and was not inhibited by a protein synthesis inhibitor cycloheximide, suggesting that these bacteria could directly activate NF-κB. STAT1, STAT3, and IFN regulatory factor-1 DNA binding was induced by both bacteria with delayed kinetics compared with NF-κB. In addition, streptococci induced the formation of IFN-α-specific transcription factor complex and IFN-stimulated gene factor-3 (ISGF3). STAT1 and STAT3 activation and ISGF3 complex formation were inhibited by cycloheximide or by neutralization with IFN-α/β-specific Abs. Streptococci were more potent than lactobacilli in inducing STAT1, ISGF3, and IFN regulatory factor-1 DNA binding. Accordingly, only streptococci induced IFN-α production. The activation of the IFN-α signaling pathway by streptococci could play a role in the pathogenesis of these bacteria. These results indicate that extracellular Gram-positive bacteria activate transcription factors involved in cytokine signaling by two mechanisms: directly, leading to NF-κB activation, and indirectly via cytokines, leading to STAT activation.

Gene expression profiling during differentiation of human monocytes to macrophages or dendritic cells

Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses. Macrophages function locally mainly, maintaining inflammatory responses in tissues, whereas DC take up microbes, mature, and migrate to local lymph nodes to present microbial antigens to naïve T cells to elicit microbe‐specific immune responses. Blood monocytes can be differentiated in vitro to macrophages or DC by GM‐CSF or GM‐CSF + IL‐4, respectively. In the present study, we performed global gene expression analyses using Affymetrix HG‐U133A Gene Chip oligonucleotide arrays during macrophage and DC differentiation. During the differentiation process, 340 and 350 genes were up‐regulated, and 190 and 240 genes were down‐regulated in macrophages and DC, respectively. There were also more that 200 genes, which were expressed differentially in fully differentiated macrophages and DC. Macrophage‐specific genes include, e.g., CD14, CD163, C5R1, and FcγR1A, and several cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin, and FcεR1A were identified as DC‐specific. Our results reveal significant differences in gene expression profiles between macrophages and DC, and these differences can partially explain the functional differences between these two important cell types.

Differential Expression of IFN Regulatory Factor 4 Gene in Human Monocyte-Derived Dendritic Cells and Macrophages

In vitro human monocyte differentiation to macrophages or dendritic cells (DCs) is driven by GM-CSF or GM-CSF and IL-4, respectively. IFN regulatory factors (IRFs), especially IRF1 and IRF8, are known to play essential roles in the development and functions of macrophages and DCs. In the present study, we performed cDNA microarray and Northern blot analyses to characterize changes in gene expression of selected genes during cytokine-stimulated differentiation of human monocytes to macrophages or DCs. The results show that the expression of IRF4 mRNA, but not of other IRFs, was specifically up-regulated during DC differentiation. No differences in IRF4 promoter histone acetylation could be found between macrophages and DCs, suggesting that the gene locus was accessible for transcription in both cell types. Computer analysis of the human IRF4 promoter revealed several putative STAT and NF-κB binding sites, as well as an IRF/Ets binding site. These sites were found to be functional in transcription factor-binding and chromatin immunoprecipitation experiments. Interestingly, Stat4 and NF-κB p50 and p65 mRNAs were expressed at higher levels in DCs as compared with macrophages, and enhanced binding of these factors to their respective IRF4 promoter elements was found in DCs. IRF4, together with PU.1, was also found to bind to the IRF/Ets response element in the IRF4 promoter, suggesting that IRF4 protein provides a positive feedback signal for its own gene expression in DCs. Our results suggest that IRF4 is likely to play an important role in myeloid DC differentiation and gene regulatory functions.

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.

KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA

Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposis sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA–mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus–host interactions during reactivation and may represent potential novel targets for therapeutic intervention.

Multiple NF-κB and IFN Regulatory Factor Family Transcription Factors Regulate CCL19 Gene Expression in Human Monocyte-Derived Dendritic Cells

CCL19 chemokine has a central role in dendritic cell (DC) biology regulating DC traffic and recruitment of naive T cells to the vicinity of activated DCs. In this study, we have analyzed the regulation of CCL19 gene expression in human monocyte-derived DCs. DCs infected with Salmonella enterica or Sendai virus produced CCL19 at late times of infection. The CCL19 promoter was identified as having two putative NF-κB binding sites and one IFN-stimulated response element (ISRE). Transcription factor binding experiments demonstrated that Salmonella or Sendai virus infection increased the binding of classical p50+p65 and alternative p52+RelB NF-κB proteins to both of the CCL19 promoter NF-κB elements. Interestingly, Salmonella or Sendai virus infection also increased the binding of multiple IFN regulatory factors (IRFs), STAT1, and STAT2, to the ISRE element. Enhanced binding of IRF1, IRF3, IRF7, and IRF9 to the CCL19 promoter ISRE site was detected in Salmonella or Sendai virus-infected cell extracts. The CCL19 promoter in a luciferase reporter construct was activated by the expression of NF-κB p50+p65 or p52+RelB dimers. IRF1, IRF3, and IRF7 proteins also activated CCL19 promoter in the presence of Sendai virus infection. CCL19 promoter constructs mutated at NF-κB and/or ISRE sites were only weakly activated. Ectopic expression of RIG-I (ΔRIG-I, CARDIF) or TLR3/4 (TRIF, MyD88, IKKε, or TBK1) signaling pathway components induced CCL19 promoter activity, suggesting that these pathways are important in CCL19 gene expression. Our experiments reveal that expression of the CCL19 gene is regulated by a combined action of several members of the NF-κB, IRF, and STAT family transcription factors.