Benjamin G Dewals

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.

ABSTRACT Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

De novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis

Neutrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly understood. We show here that apoptosis of cultured neutrophils is preceded by a substantial increase in the intracellular levels of 16 and 24 carbon atom (C16‐ and C24)‐ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonisin B2, a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C16‐ and C24‐ceramides. Moreover, fumonisin B2 significantly reduced caspase‐3, ‐8, and ‐9 activation and apoptosis in these cells. Conversely, 3‐O‐methylsphingomyelin and fantofarone, which are specific inhibitors of neutral and acid sphingomyelinases, respectively, neither inhibited C16‐ and C24‐ceramide production nor decreased the apoptosis rate in neutrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyelin. Further experiments showed that increasing endogenous C16‐ and C24‐ceramide levels by using DL‐threo‐1‐phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol and (1S,2R)‐D‐erythro‐2‐(N‐myristoylamino)‐1‐phenyl‐1‐propanol, two inhibitors of ceramide metabolism, enhances caspase‐3, ‐8, and ‐9 activity and increases neutrophil apoptosis. Similarly, apoptosis was induced rapidly when synthetic C16‐ and/or C24‐ceramides were added to neutrophil cultures. Finally, GM‐CSF, a cytokine that delays neutrophil apoptosis, abrogated C16‐ and C24‐ceramide accumulation totally in cultured neutrophils, whereas Fas ligation accelerated apoptosis in these cells without affecting de novo ceramide production. We conclude that de novo generation of C16‐ and C24‐ceramides contributes to spontaneous neutrophil apoptosis via caspase activation and that GM‐CSF exerts its antiapoptotic effects on neutrophils, at least partly through inhibition of ceramide accumulation.

IL-4Rα-independent expression of mannose receptor and Ym1 by macrophages depends on their IL-10 responsiveness.

IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation.

Bovine Herpesvirus 4 Induces Apoptosis of Human Carcinoma Cell Lines In vitro and In vivo

The idea of using oncolytic viruses for the treatment of cancers was proposed a century ago. During the last two decades, viruses able to replicate specifically in cancer cells and to induce their lysis were identified and were genetically modified to improve their viro-oncolytic properties. More recently, a new approach consisting of inducing selective apoptosis in cancer cells through viral infection has been proposed; this approach has been called viro-oncoapoptosis. In the present study, we report the property of bovine herpesvirus-4 (BoHV-4) to induce, in vitro and in vivo, apoptosis of some human carcinomas. This conclusion relies on the following observations: (a) In vitro, BoHV-4 infection induced apoptosis of A549 and OVCAR carcinoma cell lines in a time- and dose-dependent manner. (b) Apoptosis was induced by the expression of an immediate-early or an early BoHV-4 gene, but did not require viral replication. (c) Cell treatment with caspase inhibitors showed that apoptosis induced by BoHV-4 relied mainly on caspase-10 activation. (d) Infection of cocultures of A549 or OVCAR cells mixed with human 293 cells (in which BoHV-4 does not induce apoptosis) showed that BoHV-4 specifically eradicated A549 or OVCAR cancer cells from the cocultures. (e) Finally, in vivo experiments done with nude mice showed that BoHV-4 intratumoral injections reduced drastically the growth of preestablished A549 xenografts. Taken together, these results suggest that BoHV-4 may have potential as a viro-oncoapoptotic agent for the treatment of some human carcinomas. Moreover, further identification of BoHV-4 proapoptotic gene(s) and the cellular pathways targeted by this or these gene(s) could lead to the design of new cancer therapeutic strategies.

IL-4/IL-13 independent goblet cell hyperplasia in experimental helminth infections.

BackgroundIntestinal mucus production by hyperplasic goblet cells is a striking pathological feature of many parasitic helminth infections and is related to intestinal protection and worm expulsion. Induction of goblet cell hyperplasia is associated with TH2 immune responses, which in helminth infections are controlled primarily by IL-13, and also IL-4. In the study presented here we examine the goblet cell hyperplasic response to three experimental parasitic helminth infections; namely Nippostrongylus brasiliensis, Syphacia obvelata and Schistosoma mansoni.ResultsAs expected N. brasiliensis infection induced a strong goblet cell hyperplasia dependent on IL-4/IL-13/IL-4Rα expression. In contrast, and despite previously published transiently elevated IL-4/IL-13 levels, S. obvelata infections did not increase goblet cell hyperplasia in the colon. Furthermore, induction of goblet cell hyperplasia in response to S. mansoni eggs traversing the intestine was equivalent between BALB/c, IL-4/IL-13-/- and IL-4Rα-/- mice.ConclusionTogether these data demonstrate that intestinal goblet cell hyperplasia can be independent of TH2 immune responses associated with parasitic helminth infections.

Lung-resident CD4 + T cells are sufficient for IL-4Rα-dependent recall immunity to Nippostrongylus brasiliensis infection

Immunity to Nippostrongylus brasiliensis reinfection requires pulmonary CD4+ T-cell responses. We examined whether secondary lymphoid recruited or pre-existing lung CD4+ T-cell populations coordinated this immunity. To do this, we blocked T-cell egress from lymph nodes using Fingolimod (FTY720). This impaired host ability to resolve a primary infection but did not change effectiveness of recall immunity. Associated with this effective recall immunity was the expansion and T helper type 2 polarization of a pre-existing pulmonary CD4+ T-cell population. LTβR-Ig (lymphotoxin beta-receptor fusion protein)-mediated disruption of stromal cell organization of immune cells did not disrupt this recall immunity, suggesting that protection was mediated by a pulmonary interstitial residing CD4+ T-cell population. Adoptive transfer of N. brasiliensis-experienced pulmonary CD4+ T cells from FTY720-treated wild-type or T-cell interleukin (IL)-4Rα-deficient mice demonstrated protection to be IL-4Rα dependent. These results show that pre-existing CD4+ T cells can drive effective recall immunity to N. brasiliensis infection independently of T-cell recruitment from secondary lymphoid organs.

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8+ T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

IL-4Rα-responsive smooth muscle cells contribute to initiation of TH2 immunity and pulmonary pathology in Nippostrongylus brasiliensis infections

Nippostrongylus brasiliensis infections generate pulmonary pathologies that can be associated with strong TH2 polarization of the hosts immune response. We present data demonstrating N. brasiliensis-driven airway mucus production to be dependent on smooth muscle cell interleukin 4 receptor-α (IL-4Rα) responsiveness. At days 7 and 10 post infection (PI), significant airway mucus production was found in IL-4Rα−/lox control mice, whereas global knockout (IL-4Rα−/−) and smooth muscle-specific IL-4Rα-deficient mice (SM-MHCCre IL-4Rα−/lox) showed reduced airway mucus responses. Furthermore, interleukin (IL)-13 and IL-5 cytokine production in SM-MHCCre IL-4Rα−/lox mice was impaired along with a transient reduction in T-cell numbers in the lung. In vitro treatment of smooth muscle cells with secreted N. brasiliensis excretory–secretory antigen (NES) induced IL-6 production. Decreased protein kinase C (PKC)-dependent smooth muscle cell proliferation associated with cell cycle arrest was found in cells stimulated with NES. Together, these data demonstrate that both IL-4Rα and NES-driven responses by smooth muscle cells make important contributions in initiating TH2 responses against N. brasiliensis infections.

Interleukin-4-Promoted T Helper 2 Responses Enhance Nippostrongylus brasiliensis-Induced Pulmonary Pathology

ABSTRACT The role of CD4+ T-cell interleukin-4 (IL-4) receptor alpha (IL-4Rα) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4+ T-cell IL-4Rα knockout (KO) mice with the parasitic nematode Nippostrongylus brasiliensis, which induces strong host TH2 responses. Although N. brasiliensis expulsion was not affected in CD4+ T-cell IL-4Rα KO mice, the associated lung pathology was reduced. Infected CD4+ T-cell IL-4Rα KO mice showed abrogation of airway mucus production. Furthermore, CD4+ T-cell IL-4Rα KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4+ T cells was reduced in infected CD4+ T-cell IL-4Rα KO mice. Restimulation of tissue-derived CD4+ T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4+ T-cell IL-4Rα KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4+ T-cell IL-4Rα KO mice. Restimulation with N. brasiliensis antigen of total lung cell populations and populations with CD4+ T cells depleted showed that CD4+ T cells were a key TH2 cytokine source. These data demonstrated that CD4+ T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated with N. brasiliensis infections.

A gammaherpesvirus provides protection against allergic asthma by inducing the replacement of resident alveolar macrophages with regulatory monocytes

The hygiene hypothesis postulates that the recent increase in allergic diseases such as asthma and hay fever observed in Western countries is linked to reduced exposure to childhood infections. Here we investigated how infection with a gammaherpesvirus affected the subsequent development of allergic asthma. We found that murid herpesvirus 4 (MuHV-4) inhibited the development of house dust mite (HDM)-induced experimental asthma by modulating lung innate immune cells. Specifically, infection with MuHV-4 caused the replacement of resident alveolar macrophages (AMs) by monocytes with regulatory functions. Monocyte-derived AMs blocked the ability of dendritic cells to trigger a HDM-specific response by the TH2 subset of helper T cells. Our results indicate that replacement of embryonic AMs by regulatory monocytes is a major mechanism underlying the long-term training of lung immunity after infection.