Jaana Pirhonen

Role of IL-17A, IL-17F, and the IL-17 Receptor in Regulating Growth-Related Oncogene-α and Granulocyte Colony-Stimulating Factor in Bronchial Epithelium: Implications for Airway Inflammation in Cystic Fibrosis

IL-17R signaling is critical for pulmonary neutrophil recruitment and host defense against Gram-negative bacteria through the coordinated release of G-CSF and CXC chemokine elaboration. In this study, we show that IL-17R is localized to basal airway cells in human lung tissue, and functional IL-17R signaling occurs on the basolateral surface of human bronchial epithelial (HBE) cells. IL-17A and IL-17F were potent inducers of growth-related oncogene-α and G-CSF in HBE cells, and significant synergism was observed with TNF-α largely due to signaling via TNFRI. The activities of both IL-17A and IL-17F were blocked by a specific anti-IL-17R Ab, but only IL-17A was blocked with a soluble IL-17R, suggesting that cell membrane IL-17R is required for signaling by both IL-17A and IL-17F. Because IL-17A and IL-17F both regulate lung neutrophil recruitment, we measured these molecules as well as the proximal regulator IL-23p19 in the sputum of patients with cystic fibrosis (CF) undergoing pulmonary exacerbation. We found significantly elevated levels of these molecules in the sputum of patients with CF who were colonized with Pseudomonas aeruginosa at the time of pulmonary exacerbation, and the levels declined with therapy directed against P. aeruginosa. IL-23 and the downstream cytokines IL-17A and IL-17F are critical molecules for proinflammatory gene expression in HBE cells and are likely involved in the proinflammatory cytokine network involved with CF pathogenesis.

Molecular pathogenesis of influenza A virus infection and virus-induced regulation of cytokine gene expression

Despite vaccines and antiviral substances influenza still causes significant morbidity and mortality world wide. Better understanding of the molecular mechanisms of influenza virus replication, pathogenesis and host immune responses is required for the development of more efficient means of prevention and treatment of influenza. Influenza A virus, which replicates in epithelial cells and leukocytes, regulates host cell transcriptional and translational systems and activates, as well as downregulates apoptotic pathways. Influenza A virus infection results in the production of chemotactic (RANTES, MIP-1 alpha, MCP-1, MCP-3, and IP-10), pro-inflammatory (IL-1 beta, IL-6, IL-18, and TNF-alpha), and antiviral (IFN-alpha/beta) cytokines. Cytokine gene expression is associated with the activation of NF-kappa B, AP-1, STAT and IRF signal transducing molecules in influenza A virus-infected cells. In addition of upregulating cytokine gene expression, influenza A virus infection activates caspase-1 enzyme, which is involved in the proteolytic processing of proIL-1 beta and proIL-18 into their biologically active forms. Influenza A virus-induced IFN-alpha/beta is essential in hosts antiviral defence by activating the expression of antiviral Mx, PKR and oligoadenylate synthetase genes. IFN-alpha/beta also prolongs T cell survival, upregulates IL-12 and IL-18 receptor gene expression and together with IL-18 stimulates NK and T cell IFN-gamma production and the development of Th1-type immune response.

Inflammatory responses in influenza A virus infection.

Influenza A virus causes respiratory tract infections, which are occasionally complicated by secondary bacterial infections. Influenza A virus replicates in epithelial cells and leukocytes resulting in the production of chemokines and cytokines, which favor the extravasation of blood mononuclear cells and the development of antiviral and Th1-type immune response. Influenza A virus-infected respiratory epithelial cells produce limited amounts of chemokines (RANTES, MCP-1, IL-8) and IFN-alpha/beta, whereas monocytes/macrophages readily produce chemokines such as RANTES, MIP-1alpha, MCP-1, MCP-3, IP-10 and cytokines TNF-alpha, IL-1beta, IL-6, IL-18 and IFN-alpha/beta. The role of influenza A virus-induced inflammatory response in relation to otitis media is being discussed.

IFN-α Regulates TLR-Dependent Gene Expression of IFN-α, IFN-β, IL-28, and IL-29

Toll-like receptors (TLRs) mediate host cell activation by various microbial components. TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 are the receptors that have been associated with virus-induced immune response. We have previously reported that all these TLRs, except TLR9, are expressed at mRNA levels in human monocyte-derived macrophages. Here we have studied TLR2, TLR3, TLR4, and TLR7/8 ligand-induced IFN-α, IFN-β, IL-28, and IL-29 expression in human macrophages. IFN-α pretreatment of macrophages was required for efficient TLR3 and TLR4 agonist-induced activation of IFN-α, IFN-β, IL-28, and IL-29 genes. TLR7/8 agonist weakly activated IFN-α, IFN-β, IL-28, and IL-29 genes, whereas TLR2 agonist was not able to activate these genes. IFN-α enhanced TLR responsiveness in macrophages by up-regulating the expression of TLR3, TLR4, and TLR7. IFN-α also enhanced the expression of TLR signaling molecules MyD88, TIR domain-containing adaptor inducing IFN-β, IκB kinase-ε, receptor interacting protein 1, and IFN regulatory factor 7. Furthermore, the activation of transcription factor IFN regulatory factor 3 by TLR3 and TLR4 agonists was dependent on IFN-α pretreatment. In conclusion, our results suggest that IFN-α sensitizes cells to microbial recognition by up-regulating the expression of several TLRs as well as adapter molecules and kinases involved in TLR signaling.

Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most efficient antigen‐presenting cells and thus, have a major role in regulating host immune responses. In the present study, we have analyzed the ability of Gram‐positie, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte‐derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)‐2 and IL‐12 production at mRNA and protein levels. In addition, IL‐23 and IL‐27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus‐stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL‐2 or IL‐12 family cytokines was detected. Bacteria‐induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat‐inactivated. Our results show that human monocyte‐derived DCs respond differently to different Gram‐positive bacteria. Although pathogenic S. pyogenes induced a strong Th1‐type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi‐mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.

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.

Regulation of Virus-Induced IL-12 and IL-23 Expression in Human Macrophages

IL-23 is a novel cytokine that promotes the proliferation of naive and memory T cells and stimulates their IFN-γ production. Besides functional similarities, IL-23 bears structural resemblance to IL-12. Biologically active IL-23 is a heterodimer whose p40 subunit is identical to IL-12p40 while its p19 subunit is distantly related to IL-12p35. In the present study we demonstrate that human monocyte-derived macrophages are able to produce IL-23 in response to virus infection. Sendai virus stimulates the expression of p19 and p40 mRNAs in macrophages. Furthermore, it enhances p35 mRNA expression and the production of IL-12. Influenza A virus, in contrast, fails to stimulate IL-12 or IL-23 expression in macrophages. IL-12 and IL-23 contribute to the IFN-γ-inducing activity that cell culture supernatant from Sendai virus-infected macrophages show in NK-92 cells. The induction of IFN-γ production occurs in concert with IFN-αβ and IL-18, which are also secreted from the virus-infected cells. The IFN-γ-inducing activity is inhibited by IL-4, which down-regulates the transcription of p19 and p40 genes and the secretion of IFN-αβ, IL-12, and IL-18. IFN-γ, in contrast, up-regulates the p19 and p40 mRNA expression in Sendai virus infection. Thus, IL-4 and IFN-γ serve as opposing factors in the regulation of IFN-γ-inducing cytokines, including IL-23, in macrophages.

Tumor Necrosis Factor Alpha Enhances Influenza A Virus-Induced Expression of Antiviral Cytokines by Activating RIG-I Gene Expression

ABSTRACT Epithelial cells of the lung are the primary targets for respiratory viruses. Virus-carried single-stranded RNA (ssRNA) can activate Toll-like receptors (TLRs) 7 and 8, whereas dsRNA is bound by TLR3 and a cytoplasmic RNA helicase, retinoic acid-inducible protein I (RIG-I). This recognition leads to the activation of host cell cytokine gene expression. Here we have studied the regulation of influenza A and Sendai virus-induced alpha interferon (IFN-α), IFN-β, interleukin-28 (IL-28), and IL-29 gene expression in human lung A549 epithelial cells. Sendai virus infection readily activated the expression of the IFN-α, IFN-β, IL-28, and IL-29 genes, whereas influenza A virus-induced activation of these genes was mainly dependent on pretreatment of A549 cells with IFN-α or tumor necrosis factor alpha (TNF-α). IFN-α and TNF-α induced the expression of the RIG-I, TLR3, MyD88, TRIF, and IRF7 genes, whereas no detectable TLR7 and TLR8 was seen in A549 cells. TNF-α also strongly enhanced IKKε mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (ΔRIG-I) or IKKε, but not that of TLR3, enhanced the expression of the IFN-β, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-β promoter activity in TNF-α-pretreated cells. In conclusion, IFN-α and TNF-α enhanced the expression of the components of TLR and RIG-I signaling pathways, but RIG-I was identified as the central regulator of influenza A virus-induced expression of antiviral cytokines in human lung epithelial cells.

IFN-α regulates Toll-like receptor-mediated IL-27 gene expression in human macrophages

IL‐27 is a novel member of the IL‐12 cytokine family. IL‐27 has pro‐ and anti‐inflammatory properties, and it controls the responses of adaptive immunity. It promotes the differentiation of naïve Th cells and suppresses the effector functions of Th17 cells. Biologically active IL‐27 is a heterodimer composed of EBV‐induced gene 3 (EBI3) and p28 proteins. We report that TLR‐dependent expression of IL‐27 in human macrophages is mediated by IFN‐α. Stimulation of macrophages with agonists for TLR3 {polyinosinic:polycytidylic acid [poly(I:C)]}, TLR4 (LPS), or TLR7/8 (R848) results in concurrent expression of EBI3 and p28. The p28 expression is inhibited with neutralizing anti‐IFN‐α antibodies. Unlike poly(I:C), LPS, and R848, TLR2 agonist (S)‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐N‐palmitoyl‐(R)‐Cys‐(S)‐Ser(S)‐Lys4‐OH trihydrochloride does not stimulate macrophages to produce IFN‐α, and therefore, it is not able to turn on the expression of p28. There is an IFN‐stimulated response element (ISRE) in the p28 gene promoter. IFN‐α enhances the expression of IFN regulatory factor 1 (IRF‐1) in macrophages and induces binding of IRF‐1 to the p28 ISRE site. The data provide a mechanistic basis for the IFN‐α‐mediated activation of IL‐27. The data emphasize a role of IFN‐α in immune responses, which rely on the recognition of pathogens by TLRs.

Virus infection induces proteolytic processing of IL-18 in human macrophages via caspase-1 and caspase-3 activation

There is increasing evidence that IL‐18 is a key pro‐inflammatory cytokine and an important mediator of Th1 immune response. The main source of IL‐18 is macrophage‐like cells. In the present study we have investigated IL‐18 protein expression in primary human macrophages in response to influenza A and Sendai virus infections. Macrophages constitutively expressed proIL‐18 but produced biologically active IL‐18 only after virus infection. The IL‐18 release was due to virus infection‐induced proteolytic processing of 24‐kDa proIL‐18 into its mature 18‐kDa form. ProIL‐18 processing required active caspase‐1 enzyme and the release of mature IL‐18 was blocked with a caspase‐1‐specific inhibitor. Caspase‐3 inhibitor also reduced IL‐18 production in response to virus infection. Inactive proforms of caspase‐1 and caspase‐3 were basally expressed in macrophages, and virus infection induced the cleavage of procaspases into their mature forms. Besides increasing the expression of caspase proteins, virus infection enhanced caspase mRNA expression in macrophages. The enhancement of caspase gene expression was abrogated by anti‐IFN‐α antibody. Furthermore, IFN‐α and IFN‐γ could induce caspase gene expression. These results imply that interferons are involved in virus‐induced caspase activation that leads to proIL‐18 processing and subsequent release of mature IL‐18.