Søren C. Mogensen

Viral activation of macrophages through TLR-dependent and -independent pathways.

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-αβ expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-β is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-α was totally blocked in this cell line whereas IFN-αβ expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-α and RANTES expression dramatically whereas IFN-αβ induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-αβ induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-αβ on infection.

Herpes Simplex Virus Selectively Induces Expression of the CC Chemokine RANTES/CCL5 in Macrophages through a Mechanism Dependent on PKR and ICP0

ABSTRACT Recruitment of leukocytes is essential for eventual control of virus infections. Macrophages represent a leukocyte population involved in the first line of defense against many infections, including herpes simplex virus (HSV) infection. Through presentation of antigens to T cells and production of cytokines and chemokines, macrophages also constitute an important link between the innate and adaptive immune systems. Here, we have investigated the chemokine expression profile of macrophages after HSV infection and the virus-cell interactions involved. By reverse transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13 was also induced in peritoneal cells. Twenty-six other chemokines tested were not affected. Accumulation of RANTES mRNA was detectable after 5 h of infection, was sensitive to UV irradiation of the virus, and was preceded by accumulation of viral immediate-early mRNA and proteins. The viral components responsible for initiation of RANTES expression were examined with virus mutants and RAW 264.7 macrophage-like cells expressing a dominant negative mutant of the double-stranded-RNA-activated protein kinase (PKR). The PKR mutant cell line displayed reduced constitutive and HSV-inducible RANTES expression compared to the control cell line. HSV-1 mutants deficient in genes encoding the immediate-early proteins ICP4, ICP22, and ICP27 remained fully capable of inducing RANTES expression in macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to induce RANTES expression was compromised. Thus, HSV selectively induces expression of RANTES in macrophages through a mechanism dependent on cellular PKR and viral ICP0.

Herpes simplex virus type 2 induces secretion of IL-12 by macrophages through a mechanism involving NF-kappaB.

Interleukin (IL)-12 is an important proinflammatory and immunoregulatory cytokine expressed primarily by macrophages. Although IL-12 appears to be essential for clearance of many bacterial and parasitic infections, only little is known about the production and regulation of this cytokine during viral infections. In this study we have shown that infection of mouse macrophages with herpes simplex virus type 2 (HSV-2) induces secretion of the p40 subunit of IL-12, and this induction was synergistically enhanced by interferon (IFN)-γ. The production of IL-12 p40 was accompanied by production of bioactive IL-12 p70, since HSV-2-induced IFN-γ secretion was blocked by neutralizing antibodies against IL-12. The IL-12-inducing effect of HSV-2 was abrogated when virus infectivity was destroyed by heat or UV irradiation, indicating that a functional viral genome is required and that interaction of viral glycoproteins with cellular receptors is not sufficient. Production of IL-12 p40 was transcriptionally regulated and required de novo protein synthesis. Although IFN-α, IL-1β and tumour necrosis factor-α marginally influenced IL-12 production, they did not seem to constitute the endogenous factor(s) responsible for the effect of the virus infection. HSV-2 infection induced nuclear-binding activity to the κB halfsite of the IL-12 p40 promoter, and inhibitors of nuclear factor (NF)-κB activation significantly reduced IL-12 p40 production in infected cells. Collectively our data show that HSV-2 infection of murine macrophages induces production of IL-12 through a mechanism requiring intermediary synthesis of viral or host proteins and involving activation of NF-κB.

Sterility of the uterine cavity

In a prospective open study the sterility of the uterine cavity was evaluated in 99 women admitted for hysterectomy. The indications for hysterectomy were in most cases persistent irregular vaginal bleeding and fibromyomas of the uterus. Samples for both aerobic and anaerobic bacteria, Chlamydia trachomatis, yeasts and viruses were taken preoperatively from the apex of the vagina and cervical of. Immediately after hysterectomy the uterus was opened under sterile conditions and samples obtained from the isthmus and fundus of the uterine cavity for microbiological examination. Wet smears were taken from the same sites.

Expression of TNF-alpha by herpes simplex virus-infected macrophages is regulated by a dual mechanism: transcriptional regulation by NF-kappa B and activating transcription factor 2/Jun and translational regulation through the AU-rich region of the 3' untranslated region.

Here we have investigated the regulation of TNF-α expression in macrophages during HSV-2 infection. Despite a low basal level of TNF-α mRNA present in resting macrophages, no TNF-α protein is detectable. HSV-2 infection marginally increases the level of TNF-α mRNA and protein in resting macrophages, whereas a strong increase is observed in IFN-γ-activated cells infected with the virus. By reporter gene assay it was found that HSV infection augments TNF-α promoter activity. Moreover, treatment of the cells with actinomycin D, which totally blocked mRNA synthesis, only partially prevented accumulation of TNF-α protein, indicating that the infection lifts a block on translation of TNF-α mRNA. EMSA analysis showed that specific binding to the κB#3 site of the murine TNF-α promoter was induced within 1 h after infection and persisted beyond 5 h where TNF-α expression is down-modulated. Binding to the cAMP responsive element site was also induced but more transiently with kinetics closely following activation of the TNF-α promoter. Inhibitors against either NF-κB activation or the activating transcription factor 2 kinase p38 abrogated TNF-α expression, showing a requirement for both signals for activation of the promoter. This observation was corroborated by reporter gene assays. As to the translational regulation of TNF-α, the AU-rich sequence in the 3′ untranslated region of the mRNA was found to be responsible for this control because deletion of this region renders mRNA constitutively translationable. These results show that TNF-α production is induced by HSV-2 in macrophages through both transcriptional and translational regulation.

Herpes simplex virus type 2 synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha.

We have analysed the ability of herpes simplex virus type 2 (HSV-2) to induce nitric oxide (NO) production in resting BALB/c mouse peritoneal macrophages. In most experiments, macrophages produced very small amounts of NO upon infection with HSV-2. Mock virus preparations did not induce NO production, and virus inactivation experiments showed that infectious virus was required. Since interferon-gamma (IFN-gamma) is the prototype cytokine that is able to induce significant NO production in macrophages, we found it of interest to examine the influence of HSV-2 infection on the IFN-gamma-induced NO production. The virus exerted a synergistic effect on the IFN-gamma-induced NO release, which was accompanied by induction of the iNOS-gene as revealed by RT-PCR. This effect was largely dependent on the presence of infectious virus particles, since only a minor effect was seen with mock virus and inactivated virus preparations. From experiments with neutralizing antibodies to tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha/beta it was concluded that the synergistic effect is dependent on autocrine secretion of TNF-alpha, which acts as a second signal and synergizes with IFN-gamma in NO production.

CD4+ CD8+ (thymocyte-like) T lymphocytes present in blood and skin from patients with atopic dermatitis suggest immune dysregulation.

Background Atopic dermatitis (AD) is a chronic inflammatory skin disease expressed early in life. Disease development is primarily determined by as yet unknown genetic factors, leading to the accumulation of activated T lymphocytes in the skin.Objectives To investigate the nature of these T cells.Methods T‐cell lines could be established from AD skin biopsies, but not from normal skin or AD peripheral blood, when placed in RPMI 1640 medium with 10% human AB serum, antibiotics, and the T‐lymphocyte growth factors interleukins 2 and 4. The cell lines were subjected to phenotypic analysis using a fluorescence‐activated cell sorter and compared with lymphocytes from AD and normal control peripheral blood.Results T‐cell lines from 22 of 24 consecutive skin biopsies taken from 24 adult patients with AD were established. All cells were T lymphocytes expressing several activation markers. A significant proportion of the lymphocytes had stable expression of a CD4+ CD8+ phenotype (26% ± 6%; mean ± SEM). Such double‐positive T lymphocytes are normally only seen in the thymus and not in the peripheral immune system. CD4+ CD8+ cells in peripheral blood of the patients (12·5% ± 3·3%) were also detected.Conclusions We suggest that a basic pathophysiological change in AD may be a faulty maturation of the T‐lymphocyte system, leading to skin inflammation with CD4+ CD8+ T lymphocytes resembling immature T cells. This is likely to lead to skewing of many immune reactions in the patients.

Virus-Cell Interactions Regulating Induction of Tumor Necrosis Factor Alpha Production in Macrophages Infected with Herpes Simplex Virus

ABSTRACT Macrophages respond to virus infections by rapidly secreting proinflammatory cytokines, which play an important role in the first line of defense. Tumor necrosis factor alpha (TNF-α) is one of the major macrophage-produced cytokines. In this study we have investigated the virus-cell interactions responsible for induction of TNF-α expression in herpes simplex virus (HSV)-infected macrophages. Both HSV type 1 (HSV-1) and HSV-2 induced TNF-α expression in macrophages activated with gamma interferon (IFN-γ). This induction was to some extent sensitive to UV treatment of the virus. Virus particles unable to enter the cells displayed reduced capacity to stimulate TNF-α expression but retained a significant portion which was abolished by HSV-specific antibodies. Recombinant HSV-1 glycoprotein D was able to trigger TNF-α secretion in concert with IFN-γ. Sugar moieties of HSV glycoproteins have been reported to be involved in induction of IFN-α but did not contribute to TNF-α expression in macrophages. Moreover, the entry-dependent portion of the TNF-α induction was investigated with HSV-1 mutants and found to be independent of the tegument proteins VP16 and UL13 and partly dependent on nuclear translocation of the viral DNA. Finally, we found that macrophages expressing an inactive mutant of the double-stranded RNA (dsRNA)-activated protein kinase (PKR) produced less TNF-α in response to infectious HSV infection than the empty-vector control cell line but displayed the same responsiveness to UV-inactivated virus. These results indicate that HSV induces TNF-α expression in macrophages through mechanisms involving (i) viral glycoproteins, (ii) early postentry events occurring prior to nuclear translocation of viral DNA, and (iii) viral dsRNA-PKR.

Effect of IL-4 and IL-13 on IFN-γ-induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2

Interleukin (IL)‐4 and IL‐13 share a wide range of activities. Prominent among these is the ability to antagonize many interferon (IFN)‐γ‐induced activities. Here we demonstrate that IL‐4 and IL‐13 totally abrogate IFN‐γ‐induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA and protein synthesis in a murine macrophage cell line. IFN‐γ‐treated cells infected with herpes simplex virus type 2 (HSV‐2) or costimulated with tumor necrosis factor (TNF)‐α showed an enhanced reactivity, which was only partially reduced by IL‐4/13. The results indicate that IL‐4 and IL‐13 function by intervening with a step prior to iNOS transcription by antagonizing IFN‐γ‐induced signal(s) without counteracting synergistic virus‐ or TNF‐α‐induced signals. The beneficial effect of a sustained NO production in foci of virus infection is suggested.

Acyclovir given as prophylaxis against oral ulcers in acute myeloid leukaemia: randomised, double blind, placebo controlled trial.

Abstract Objectives: To evaluate (a) the prophylactic effect of the antiherpetic drug acyclovir on oral ulcers in patients with acute myeloid leukaemia receiving remission induction chemotherapy and thus (b), indirectly, the role of herpes simplex virus in the aetiology of these ulcers. Design: Randomised, double blind, placebo controlled trial. Subjects: 74 herpes simplex virus seropositive patients aged 18-84. Thirty seven patients received acyclovir (800 mg by mouth daily) and 37 placebo. The patients were examined daily for 28 days. Main outcome measures: Occurrence of herpes labialis, intraoral ulcers, and acute necrotising ulcerative gingivitis. Results: The two populations were comparable in age, sex, type of antineoplastic treatment, and history of herpes labialis. Acute oral infections occurred in 25 of the acyclovir treated patients and 36 of the placebo treated patients (relative risk 0.69 (95% confidence interval 0.55 to 0.87)). This difference was due to a reduction in the incidence of herpes labialis (one case versus eight cases; relative risk 0.13 (0.02 to 0.95)), intraoral ulcers excluding the soft palate (one case versus 13 cases; relative risk 0.08 (0.01 to 0.56)), and acute necrotising ulcerative gingivitis (one case versus eight cases; relative risk 0.13 (0.02 to 0.95)). However, ulcers on the soft palate were diagnosed with similar frequency in the two groups. Isolation of herpes simplex virus type 1 in saliva was reduced from 15 cases in the placebo group to one case in the acyclovir group (relative risk 0.07 (0.01 to 0.48)). Conclusion: Intraoral ulcers excluding the soft palate are most often due to infection with herpes simplex virus, whereas ulcers on the soft palate have a non-herpetic aetiology. The findings suggest that acute necrotising ulcerative gingivitis may also be due to herpes simplex virus. Prophylaxis with acyclovir should be considered for patients with acute myeloid leukaemia during remission induction therapy. Key messages Key messages Oral ulcers were found in more than half of patients with acute myeloid leukaemia during remission induction therapy The incidence of herpes labialis, intraoral ulcers outside the soft palate, and acute necrotising ulcerative gingivitis was greatly reduced by prophylaxis with oral acyclovir (800 mg by mouth daily) during remission induction therapy Intraoral ulcers on the soft palate were not associated with herpes virus infection