Nicholas Megjugorac

Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells

Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte‐derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV‐1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV‐1. All three populations constitutively expressed most of the IRF genes. However, real‐time RT‐PCR demonstrated increased levels of IRF‐7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF‐7 protein than the other populations while IRF‐3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus‐stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus‐dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN‐α production due to the high levels of constitutively expressed IRF‐7.

Regulation of IFN Regulatory Factor-7 and IFN-α Production by Enveloped Virus and Lipopolysaccharide in Human Plasmacytoid Dendritic Cells

Human plasmacytoid dendritic cells (PDC) are a major source of IFN-α upon exposure to enveloped viruses and TLR-7 and TLR-9 ligands. Although IFN regulatory factor-7 (IRF-7) is known to play an essential role in virus-activated transcription of IFN-α genes, the molecular mechanisms of IFN-α production in human PDC remain poorly understood. We and others have recently reported high constitutive levels of IRF-7 expression in PDC as compared with other PBMC. In this study, we demonstrate that both LPS and HSV up-regulate the expression of IRF-7 in PDC, and that this enhancement of IRF-7 is dependent on NF-κB activation. The NF-κB inhibitors MG132 and pyrrolidinedithiocarbamate efficiently inhibited the induction of IRF-7 by HSV or LPS, and also down-regulated the constitutive expression of IRF-7 in PDC and blocked the HSV-induced production of IFN-α. In addition, we found that nuclear translocation of IRF-7 occurred rapidly in response to HSV stimulation, but not in response to LPS, which is consistent with the stimulation of IFN-α production by virus and not by LPS. Although LPS by itself was not able to induce IFN-α production, it led to rapid up-regulation of TLR-4 on PDC and increased the magnitude and accelerated the kinetics of HSV-induced IFN-α production in PDC, providing a mechanism that might be operative in a scenario of mixed infection. In contrast to the current concept of IFN-α regulation established in cell lines, this study strongly supports the immediate availability of high constitutive levels of IRF-7 expression in PDC, and suggests an activation required for IRF-7 that contributes to IFN-α production in virus-stimulated PDC.

IFN-λ1 (IL-29) inhibits GATA3 expression and suppresses Th2 responses in human naive and memory T cells

IFN-lambda1 (IL-29) plays a novel, emerging role in the inhibition of human Th2 responses. Here, we demonstrate that both naive and memory human CD4(+) T cells express mRNA for the IFN-lambda1-specific receptor, IL-28Ralpha, and are responsive to IFN-lambda1. Expression of Th2 cytokines (IL-4 and IL-13) was suppressed in naive and memory CD4(+) T cells by IFN-lambda1, without affecting their proliferation. Further, acquisition of IL-4Ralpha expression after stimulation was inhibited by IFN-lambda1, as was GATA3 expression. Finally, IFN-lambda1 diminished the change in cell-surface phenotype that accompanies differentiation of central memory T cells into effector memory T cells. Taken together, our data describe unique immunomodulatory effects of IFN-lambda1 and identify novel mechanisms for the reduction of existing Th2 responses and the regulation of new ones, in circulating naive and memory CD4(+) T cells.

Receptor Cross-Linking on Human Plasmacytoid Dendritic Cells Leads to the Regulation of IFN-α Production

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-α in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-α production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-α production at the level of IRF-7, while the decrease in IFN-α production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.

Modulation of human plasmacytoid DC function by IFN-λ1 (IL-29)

The type III family of IFNs displays immunomodulatory and antiviral activity. Each member (IFN‐λ1, −2, and −3) signals through the same heterodimeric receptor complex, which consists of the binding and signaling subunit (IL‐28Rα) plus the IL‐10Rβ chain. Although the receptor has a wide tissue distribution, the direct effects of IFN‐λ on various immune cell subsets have not been fully characterized. We have identified high levels of IL‐28Rα mRNA in pDC from peripheral blood and hypothesized that IFN‐λ plays an important role in pDC maturation and development. We show that stimulation of pDC with HSV or Imiquimod causes an increase in IL‐28Rα mRNA. In these cells, IFN‐λ1 alters expression of the costimulatory molecules CD80 and ICOS‐L and synergizes with IFN‐α to up‐regulate CD83. In addition, IFN‐λ1 has a variable effect on the homing molecule expression of pDC and mDC. IFN‐λ1‐treated pDC display a marked difference in their ability to stimulate production of the signature cytokines IL‐13, IFN‐γ, and IL‐10 in a MLR. This work characterizes the variable effects of IFN‐λ on DC surface molecule expression and identifies a role in pDC activation and immunostimulatory potential.

Modulation of human β-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD‐2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD‐1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD‐1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD‐1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV‐1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD‐1 mRNA levels, as measured by qRT‐PCR at 3 h following infection. A similar inhibition occurred with HSV‐1 challenge of human gingival epithelial cells. Studies with HSV‐1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD‐1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD‐1(−/−) mice with mouse‐adapted HK18 (300 PFU/mouse). mBD‐1(−/−) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD‐1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD‐1(−/−) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD‐1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.

IL-4 enhances IFN-λ1 (IL-29) production by plasmacytoid DC, via monocyte secretion of IL-1Ra

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]), IFN-lambda2 (IL-28A), and IFN-lambda3 (IL-28B), each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently, it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here, we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However, pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments, we showed that IL-4-responsive monocytes are an intermediary cell, responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus, our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function, and suggests an expanded immunomodulatory role for Th2-associated cytokines.

The lambda interferons: guardians of the immune-epithelial interface and the T-helper 2 response.

The type-III interferons (IFNs) are the most recently discovered IFNs in the human immune system and have important, but as yet poorly characterized, functions in innate and adaptive immunity that complement their antiviral functions. It is now becoming clear that these type-III IFNs have a functional niche where epithelial surfaces interact with the adaptive immune system, that their antiviral capability is not as highly developed as that of the type-I IFNs, and that they have their own profile of immunomodulatory functions; specifically, they are key modulators of the T-helper (Th)2 response.

Image-Based Study of Interferongenic Interactions between Plasmacytoid Dendritic Cells and HSV-Infected Monocyte-Derived Dendritic Cells

Plasmacytoid dendritic cells (pDC) are well-known for their ability to produce large quantities of interferon-α (IFN-α) in response to viruses. In addition, pDC produce IFN-α in response to HSV-infected cells. We demonstrate that both tonsil and PBMC contain pDC that respond to stimulation with HSV either in suspension or in tonsil tissue-fragment culture. We hypothesized that other DC subsets acquire virus in the periphery and deliver the interferongenic signals to the pDC in the draining lymphoid tissue. As a model for pDC/myeloid DC interaction, we studied the interaction of pDC derived from blood with HSV-infected and uninfected monocyte derived dendritic cells (MDDC). Infected, but not uninfected, MDDC induced IFN-α in pDC. To further study pDC/infected MDDC interactions, we labeled MDDC with fluorescent cell trackers PKH67 or CFSE prior to infection with HSV and co-cultured with pDC. Cells were then analyzed using conventional and imaging flow cytometry. In addition, we infected MDDC with a GFP-expressing HSV prior to co-culture with pDC. Using traditional flow cytometry, we observed that pDC became fluorescent after co-incubation with uninfected or infected, fluorescently labeled MDDC, indicating that MDDC transferred fluorescent protein and membrane to pDC. By imaging flow cytometry, we observed formation of conjugates between pDC and MDDC as well as transfer and internalization of cellular components from the labeled MDDC by pDC, with preferential uptake from, and association with, infected vs. uninfected MDDC. These studies demonstrate that MDDC infected with HSV are able to stimulate IFN-α and chemokine production by pDC through the transfer of cellular materials from the HSV-infected MDDC to the pDC. Together, these observations indicate that heterogeneous populations of DC interact to generate an effective IFN-α response.

Immune-related Serum miRNAs Are Differentially Expressed in IBD Patients Compared to Healthy Controls: P-104

small bowel. Mean total LS at very early postoperative period was 428.3 (range: 8-4264). Based on total LS, 78% (14/18) of patients corresponded to mild to severe active disease (LS>135). These lesions were evenly distributed in all segments of the remaining small intestine. Mean LS of proximal, mid and distal tertile was 286.6 6 994.5, 83.0 6 133.2 and 146.7 6 170.3, respectively. Ulcers on ileo-colonic and ileo-ileal anastomosis were observed in 83% of them. At 6-8 months after surgery, 13 patients underwent follow-up CE. All 13 patients were in clinical remission at follow-up period. Sixty-nine% of patients was on scheduled infliximab within one month of surgery. Endoscopic progressive recurrence which defined as an increase of total LS more than 100 points in comparison to pervious finding was observed in 38% of patients. No patients showed complications related to CE. CONCLUSION(S): Many residual small bowel inflammatory lesions were observed by CE at very early postoperative period in patients with CD after radical or curative resection. Repeated CE may be useful technique for postoperative assessment of endoscopic recurrence at regularly follow-up in CD.