Rotavirus epidemiology 5-6 years after universal rotavirus vaccination: persistent rotavirus activity in older children and elderly.

BACKGROUND: Rotavirus (RV) vaccination using RotaTeq® vaccine exclusively was introduced into Finnish National Immunization Program (NIP) in 2009, and soon reached high (≥90%) coverage. Since mid-2013, all stool samples from laboratory diagnosed cases of RV gastroenteritis in the entire country have been typed. METHODS: 364 RV positive stool samples collected from clinical laboratories over a 2-year period were G- and P-typed using RT-PCR, and the results were confirmed by sequencing. In addition, the genome segment encoding for VP6 was sequenced to distinguish between wild-type and vaccine origin (bovine) RVs. RESULTS: RV winter epidemic seasons 2013-2014 and 2014-2015 lasted until July each. The age distribution of RV cases showed two unusual clusters: one in children 6-16 years of age, too old to have been vaccinated in NIP, and the other in elderly over 70 years of age. In children, diverse genotypes were observed without any obvious predominance. The most common ones were G1P[8] (30.0%), G2P[4] (22.4%), G9P[8] (15.8%), G3P[8] (12.2%) and G4P[8] (11.2%). The genotype distribution was not different among vaccinated and unvaccinated children. Most cases in the elderly were associated with G2P[4]. CONCLUSIONS: Even at high vaccine coverage and high effectiveness of RV vaccine, RV activity continues to persist, particularly in unvaccinated older children. RV genotypes show greater diversity than before RV vaccinations. We conclude that RV disease can be controlled but not eliminated by vaccinations. Herd-protection in long-term follow-up may be less than at the start of RV vaccinations.

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-1ß 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.

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.

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-alpha/beta]) and type III (IFN-lambda1 to -lambda3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-alpha) 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-alpha and IFN-beta inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-lambda3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-lambda3 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.

IFN-alpha 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-alpha. 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-alpha 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-alpha, 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-alpha 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-alpha-mediated activation of IL-27. The data emphasize a role of IFN-alpha in immune responses, which rely on the recognition of pathogens by TLRs.

Interleukin 23 in acute inflammatory demyelination of the peripheral nerve.

BACKGROUND: Interleukin (IL) 23, a newly identified heterodimeric proinflammatory cytokine and a novel IL-12 family member comprising the p40 subunit of IL-12 but a different p19 subunit, has been reported to preferentially act on memory T cells and play an important role during cellular immune responses. Recent evidence suggests that IL-23 rather than IL-12 is critically involved in the pathogenesis of various immune-mediated disorders. OBJECTIVE: To determine the role of IL-23p19 during the course of acute immune-mediated demyelinating diseases of the peripheral nervous system. DESIGN: The sequential RNA expression of IL-23p19 in sciatic nerves from rats with experimental autoimmune neuritis, an animal model of the human Guillain-Barré syndrome (GBS), was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction. Expression and distribution patterns of IL-23p19 protein were studied in sural nerve biopsies and cerebrospinal fluid samples from 5 patients with classical Guillain-Barré syndrome and 5 controls with noninflammatory neuropathies using immunohistochemistry and immunoblotting, respectively. RESULTS: We found IL-23p19 RNA to be up-regulated prior to the onset of first clinical symptoms with peak expression levels preceding maximum disease severity during experimental autoimmune neuritis. In patients, IL-23p19 protein was detectable in cerebrospinal fluid samples from patients with Guillain-Barré syndrome, and endoneurial macrophages were identified as the cellular source of IL-23p19 in sural nerve biopsies. CONCLUSION: Our present data indicate that IL-23 may play an important role during the early effector phase in immune-mediated demyelination of the peripheral nerve.

Tumor necrosis factor alpha enhances influenza A virus-induced expression of antiviral cytokines by activating RIG-I gene expression.

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-alpha), IFN-beta, 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-alpha, IFN-beta, 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-alpha or tumor necrosis factor alpha (TNF-alpha). IFN-alpha and TNF-alpha 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-alpha also strongly enhanced IKK epsilon mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (deltaRIG-I) or IKK epsilon, but not that of TLR3, enhanced the expression of the IFN-beta, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-beta promoter activity in TNF-alpha-pretreated cells. In conclusion, IFN-alpha and TNF-alpha 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.

Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth-related oncogene-alpha 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-alpha and G-CSF in HBE cells, and significant synergism was observed with TNF-alpha 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.

IFN-alpha regulates TLR-dependent gene expression of IFN-alpha, IFN-beta, 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-alpha, IFN-beta, IL-28, and IL-29 expression in human macrophages. IFN-alpha pretreatment of macrophages was required for efficient TLR3 and TLR4 agonist-induced activation of IFN-alpha, IFN-beta, IL-28, and IL-29 genes. TLR7/8 agonist weakly activated IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas TLR2 agonist was not able to activate these genes. IFN-alpha enhanced TLR responsiveness in macrophages by up-regulating the expression of TLR3, TLR4, and TLR7. IFN-alpha also enhanced the expression of TLR signaling molecules MyD88, TIR domain-containing adaptor inducing IFN-beta, IkappaB kinase-epsilon, 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-alpha pretreatment. In conclusion, our results suggest that IFN-alpha sensitizes cells to microbial recognition by up-regulating the expression of several TLRs as well as adapter molecules and kinases involved in TLR signaling.

Cytokine and contact-dependent activation of natural killer cells by influenza A or Sendai virus-infected macrophages.

NK cells participate in innate immune responses by secreting gamma interferon (IFN-gamma) and by destroying virus-infected cells. Here the interaction between influenza A or Sendai virus-infected macrophages and NK cells has been studied. A rapid, cell-cell contact-dependent production of IFN-gamma from NK cells cultured with virus-infected macrophages was observed. Expression of the MHC class I-related chain B (MICB) gene, a ligand for NK cell-activating receptor NKG2D, was upregulated in virus-infected macrophages suggesting a role for MICB in the activation of the IFN-gamma gene in NK cells. IL12Rbeta2, IL18R and T-bet mRNA synthesis was enhanced in NK cells cultured with virus-infected macrophages. Upregulation of these genes was dependent on macrophage-derived IFN-alpha. In contrast to IL12Rbeta2, expression of WSX-1/TCCR, a receptor for IL27, was reduced in NK cells in response to virus-induced IFN-alpha. In conclusion, these results show that virus-infected macrophages activate NK cells via cytokines and direct cellular interactions and further emphasize the role of IFN-alpha in the activation of innate immunity.

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-positive, 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.

Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells.

IL-12 p40-related cytokines such as IL-12 p35/p40 heterodimer and IL-23 (p19/p40) are potent regulators of adaptive immune responses. Little is known, however, about the transcriptional regulation of the p40 gene in vivo. In an attempt toward this goal, we have generated transgenic mice expressing firefly luciferase under the control of the IL-12 p40 promoter. High constitutive transgene expression was found in the small intestine only, whereas little reporter gene activity was observed in other tissues. Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression of p40 mRNA as well as p40 and IL-23 p19/p40 proteins. The cells constitutively producing IL-12 p40 were identified as CD8alpha and CD11b double-negative CD11c+ lamina propria dendritic cells (LPDCs) that represent a major cell population in the lamina propria of the small intestine, but not in the colon. FISH directly demonstrated the uptake of bacteria by a subset of LPDCs in the terminal ileum that was associated with p40 expression. Furthermore, little or no p40 protein expression in LPDCs was found in the terminal ileum of germfree mice, indicating a key role of the intestinal flora for constitutive p40 expression. In addition, analysis of transgenic mice with a mutated NF-kappaB target site in the p40 promoter showed a critical role of NF-kappaB for constitutive transgene expression. Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDCs through NF-kappaB. These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses through IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn disease in this part of the gut.

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-gamma 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-gamma-inducing activity that cell culture supernatant from Sendai virus-infected macrophages show in NK-92 cells. The induction of IFN-gamma production occurs in concert with IFN-alphabeta and IL-18, which are also secreted from the virus-infected cells. The IFN-gamma-inducing activity is inhibited by IL-4, which down-regulates the transcription of p19 and p40 genes and the secretion of IFN-alphabeta, IL-12, and IL-18. IFN-gamma, in contrast, up-regulates the p19 and p40 mRNA expression in Sendai virus infection. Thus, IL-4 and IFN-gamma serve as opposing factors in the regulation of IFN-gamma-inducing cytokines, including IL-23, in macrophages.