Coxsackievirus B4 Transplacental Infection Severely Disturbs Central Tolerogenic Mechanisms in the Fetal Thymus.

Thymus plays a fundamental role in central tolerance establishment, especially during fetal life, through the generation of self-tolerant T cells. This process consists in T cells education by presenting them tissue-restricted autoantigens promiscuously expressed by thymic epithelial cells (TECs), thus preventing autoimmunity. Thymus infection by Coxsackievirus B (CV-B) during fetal life is supposed to disturb thymic functions and, hence, to be an inducing or accelerating factor in the genesis of autoimmunity. To further investigate this hypothesis, in our current study, we analyzed thymic expression of autoantigens, at the transcriptional and protein level, following in utero infection by CV-B4. mRNA expression levels of Igf2 and Myo7, major autoantigens of pancreas and heart, respectively, were analyzed in whole thymus and in enriched TECs together along with both transcription factors, Aire and Fezf2, involved in autoantigens expression in the thymus. Results show that in utero infection by CV-B4 induces a significant decrease in Igf2 and Myo7 expression at both mRNA and protein level in whole thymus and in enriched TECs as well. Moreover, a correlation between viral load and autoantigens expression can be observed in the whole thymus, indicating a direct effect of in utero infection by CV-B4 on autoantigens expression. Together, these results indicate that an in utero infection of the thymus by CV-B4 may interfere with self-tolerance establishment in TECs by decreasing autoantigen expression at both mRNA and protein level and thereby increase the risk of autoimmunity onset.

Cytotoxicity and Antiviral Activities of Haplophyllum tuberculatum Essential Oils, Pure Compounds, and Their Combinations against Coxsackievirus B3 and B4.

Haplophyllum tuberculatum is a plant commonly used in folk medicine to treat several diseases including vomiting, nausea, infections, rheumatism, and gastric pains. In the current study, H. tuberculatum essential oils, hydrosols, the pure compounds R-(+)-limonene, S-(-)-limonene, and 1-octanol, as well as their combinations R-(+)-limonene/1-octanol and S-(-)-limonene/1-octanol, were screened for their cytotoxicity on HEp-2 cells after 24, 48, and 72 h, and then tested for their activity against Coxsackievirus B3 and B4 (CV-B3 and CV-B4) at 3 different moments: addition of the plant compounds before, after, or together with virus inoculation. Results showed that the samples were more cytotoxic after 72 h than after 24 h or 48 h cell contact. However, the combinations R-(+)-limonene/1-octanol and S-(-)-limonene/1-octanol showed less effect on HEp-2 cells than pure R-(+)-limonene and S-(-)-limonene after 24 h, 48 h, and 72 h. 1-octanol exhibited the highest concentration causing 50% cytotoxicity (CC50) on HEp-2 cells after 24 h (CC50 = 93 µg/mL) and 48 h (CC50 = 83 µg/mL). The antiviral assays showed that the tested samples exhibited potent inhibition of CV-B. IC50 values ranged from 0.66 µg/mL to 28.4 µg/mL. In addition, CV-B3 was more sensitive than CV-B4. Both CV-B strains are more inhibited when cells were pretreated with the plant compounds. The hydrosols have no effect, neither on HEp-2 cells nor on the virus. 1-octanol, S-(-), and R-(+)-limonene/1-octanol had important selectivity indexes over time. Although essential oils had potent antiviral activity, they can be considered for application in the pretreatment of cells. However, 1-octanol and the combinations are within the safety limits, and thus, they can be used as an active natural antiviral agent for CV-B3 and CV-B4 inhibition.

Effect of Coxsackievirus B4 Infection on the Thymus: Elucidating Its Role in the Pathogenesis of Type 1 Diabetes.

The thymus gland is a primary lymphoid organ for T-cell development. Various viral infections can result in disturbance of thymic functions. Medullary thymic epithelial cells (mTECs) are important for the negative selection of self-reactive T-cells to ensure central tolerance. Insulin-like growth factor 2 (IGF2) is the dominant self-peptide of the insulin family expressed in mTECs and plays a crucial role in the intra-thymic programing of central tolerance to insulin-secreting islet ß-cells. Coxsackievirus B4 (CVB4) can infect and persist in the thymus of humans and mice, thus hampering the T-cell maturation and differentiation process. The modulation of IGF2 expression and protein synthesis during a CVB4 infection has been observed in vitro and in vivo in mouse models. The effect of CVB4 infections on human and mouse fetal thymus has been studied in vitro. Moreover, following the inoculation of CVB4 in pregnant mice, the thymic function in the fetus and offspring was disturbed. A defect in the intra-thymic expression of self-peptides by mTECs may be triggered by CVB4. The effects of viral infections, especially CVB4 infection, on thymic cells and functions and their possible role in the pathogenesis of type 1 diabetes (T1D) are presented.

Modulation of IGF2 Expression in the Murine Thymus and Thymic Epithelial Cells Following Coxsackievirus-B4 Infection.

Coxsackievirus B4 (CV-B4) can infect human and murine thymic epithelial cells (TECs). In a murine TEC cell line, CV-B4 can downregulate the transcription of the insulin-like growth factor 2 (Igf2) gene coding for the self-peptide of the insulin family. In this study, we show that CV-B4 infections of a murine TEC cell line decreased Igf2 P3 promoter activity by targeting a region near the transcription start site; however, the stability of Igf2 transcripts remained unchanged, indicating a regulation of Igf2 transcription. Furthermore, CV-B4 infections decreased STAT3 phosphorylation in vitro. We also showed that mice infected with CV-B4 had an altered expression of Igf2 isoforms as detected in TECs, followed by a decrease in the pro-IGF2 precursor in the thymus. Our study sheds new light on the intrathymic regulation of Igf2 transcription during CV-B4 infections and supports the hypothesis that a viral infection can disrupt central self-tolerance to insulin by decreasing Igf2 transcription in the thymic epithelium.

Coxsackievirus B4 infection and interneuronal spread in primary cultured neurons.

Coxsackie B viruses (CV-B) are usually transmitted via the fecal-oral route and the virus gains the central nervous system (CNS) via the bloodstream. Nevertheless, other routes of spread of the virus to the CNS cannot be excluded, including the neuronal route. Neuronal cells, as well as non-neuronal cells (fibroblasts), were isolated from mice and inoculated with CV-B4 in the absence and presence of neutralizing serum. In the absence of neutralizing serum, virus titers recorded in neuron cultures and rates of infected neurons were non-significantly different compared to those recorded in fibroblast cultures. Higher cell mortality was noted among neurons than fibroblasts. The addition of neutralizing serum to neurons did not reduce significantly virus titers or rates of infected cells and cell viability was not significantly augmented, while virus titers and rates of infected fibroblasts were significantly reduced and their viability was significantly enhanced as well. Our results demonstrate the ineffectiveness of neutralizing serum to prevent neurons infection with CV-B4 which suggests a trans-synaptic transmission of CV-B4 between neurons.

Assessment of Thymic Output Dynamics After in utero Infection of Mice With Coxsackievirus B4.

The thymus is the main organ of the lymphatic system, in which T cells undergo a rigorous selection to ensure that their receptors (TCRs) will be functional and will not react against the self. Genes encoding for TCR chains are fragmented and must be rearranged by a process of somatic recombination generating TCR rearrangement excision circles (TRECs). We recently documented coxsackievirus B4 (CV-B4) infection of Swiss albino mouse thymus in the course of in utero transmission. In the current study, we intended to evaluate thymic output in this experimental model. For this purpose, pregnant Swiss albino mice were inoculated with CV-B4 at day 10 or 17 of gestation, and thymus and spleen were sampled from offspring at different time points and then subjected to quantification of TREC molecules and Ptk7 gene expression. Results showed a pronounced effect of in utero CV-B4 infection on the thymus with an increase in the cellularity and, consequently, the weight of the organ. sj and DßTREC analysis, by real-time PCR, revealed a significant decrease following CV-B4 infection compared to controls, a decrease which gets worse as time goes by, both in the thymus and in the periphery. Those observations reflect a disturbance in the export of T cells to the periphery and their accumulation within the thymus. The evaluation of Ptk7 transcripts in the thymus, for its part, showed a decrease in expression, especially following an infection at day 10 of gestation, which supports the hypothesis of T cell accumulation in a mature stage in the thymus. The various effects observed correlate either negatively or positively with the viral load in the thymus and spleen. Disruption in thymic export may indeed interfere with T cell maturation. We speculate that this may lead to a premature release of T cells and the possibility of circulating autoreactive or proliferation-impaired T cell clones.

Housekeeping Gene Expression in the Fetal and Neonatal Murine Thymus Following Coxsackievirus B4 Infection.

The thymus fulfills the role of T-cell production and differentiation. Studying transcription factors and genes involved in T-cell differentiation and maturation during the fetal and neonatal periods is very important. Nevertheless, no studies to date have been interested in evaluating the expressions of housekeeping genes as internal controls to assess the varying expressions of different genes inside this tissue during that period or in the context of viral infection. Thus, we evaluated by real-time quantitative polymerase chain reaction (qPCR) the expression of the most common internal control genes in the thymus of Swiss albino mice during the fetal and neonatal period, and following in utero infection with Coxsackievirus B4. The stability of expression of these reference genes in different samples was investigated using the geNorm application. Results demonstrated that the expression stability varied greatly between genes. Oaz1 was found to have the highest stability in different stages of development, as well as following Coxsackievirus B4 infection. The current study clearly demonstrated that Oaz1, with very stable expression levels that outperformed other tested housekeeping genes, could be used as a reference gene in the thymus and thymic epithelial cells during development and following Coxsackievirus B4 infection.

Immunopathology in the brain of mice following vertical transmission of Coxsackievirus B4.

Coxsackie B viruses (CV-B) are associated with several central nervous system (CNS) disorders. These viruses are predominantly transmitted by fecal-oral route but vertical transmission can also occur. This work attempted to study the immune response ensuing vertical transmission of CV-B to the brain, and its eventual implementation in the brain pathogenesis. To this end, pregnant Swiss albino mice were inoculated with CV-B4 E2 or with sterile medium for control animals. At different ages after birth, brains were collected and analyzed for virus infection, histopathological changes and immune response. Infectious particles were detected in offspring's brain which demonstrates vertical transmission of the virus. This infection is persistent since the long lasting detection of viral RNA in offspring's brain. Some pathological signs including meningitis, edema and accumulation of inflammatory cells within and surrounding the inflammatory areas were observed. Immunoflorescence staining unveiled the presence of T lymphocytes and microgliosis in the sites of lesion for a long period after birth. Multiplex cytokines measurement upon supernatants of in vitro mixed brain cells and extracted mononuclear cells from offspring's brain has demonstrated an elevated secretion of the pro-inflammatory cytokines TNFα, IL-6 and IFNα and the chemokines RANTES and MCP-1. Hence, vertical transmission of CV-B4 and its persistence within offspring's brain can lead to pathological features linked to increased and sustained immune response.

The presentation of neuroendocrine self-peptides in the thymus: an essential event for individual life and vertebrate survival.

Confirming Burnet's early hypothesis, elimination of self-reactive T cells in the thymus was demonstrated in the late 1980s, and an important question immediately arose about the nature of the self-peptides expressed in the thymus. Many genes encoding neuroendocrine-related and tissue-restricted antigens (TRAs) are transcribed in thymic epithelial cells (TECs). They are then processed for presentation by proteins of the major histocompatibility complex (MHC) expressed by TECs and thymic dendritic cells. MHC presentation of self-peptides in the thymus programs self-tolerance by two complementary mechanisms: (1) negative selection of self-reactive "forbidden" T cell clones starting already in fetal life, and (2) generation of self-specific thymic regulatory T lymphocytes (tTreg cells), mainly after birth. Many studies, including the discovery of the transcription factors autoimmune regulator (AIRE) and fasciculation and elongation protein zeta family zinc finger (FEZF2), have shown that a defect in thymus central self-tolerance is the earliest event promoting autoimmunity. AIRE and FEZF2 control the level of transcription of many neuroendocrine self-peptides and TRAs in the thymic epithelium. Furthermore, AIRE and FEZF2 mutations are associated with the development of autoimmunity in peripheral organs. The discovery of the intrathymic presentation of self-peptides has revolutionized our knowledge of immunology and is opening novel avenues for prevention/treatment of autoimmunity.

Coxsackievirus B4 vertical transmission in a murine model.

BACKGROUND: Life-threatening infections with type B Coxsackieviruses (CV-B) are frequently encountered among newborns and are partly attributed to vertically-transmitted virus. Our current study investigates this alternative way of contamination by CV-B, using a mouse model. METHODS: Pregnant Swiss mice were intraperitoneally inoculated with CV-B4 E2 at gestational day 10(G) or 17G. Dams and offspring were monitored for mortality and morbidity, and sampled at different time-points to document the infection and explore eventual vertical transmission. RESULTS: Inoculation at day 10G induced an important rate of abortion and a decrease in the number of delivered pups per litter, whereas inoculation at day 17G was marked by preterm delivery and significant behavioral changes in dams. Only one case of spastic paralysis and one case of pancreatitis were recorded among surviving pups. Seroneutralization revealed anti-CV-B4 neutralizing antibodies in infected dams and their partial transfer to offspring. Viral genome detection by RT-PCR and viral progeny titration in several tissues (dams' uteri, amniotic sac, amniotic fluid, placenta, umbilical cord, pancreas and heart) attested and documented CV-B4 vertical transmission to the majority of analyzed offspring. Virus detection in fetuses suggests transplacental transmission, but perinatal transmission during delivery could be also suggested. Vertically transmitted CV-B might even persist since prolonged viral RNA detection was noticed in the pancreas and heart from offspring born to dams inoculated at day 17G. CONCLUSION: This model of CV-B4 vertical transmission in mice, in addition to allow a better understanding of CV-B infections in fetuses and newborns, constitutes a useful tool to investigate the pathogenesis of CV-B associated chronic diseases.

Anti-coxsackievirus B4 (CV-B4) enhancing activity of serum associated with increased viral load and pathology in mice reinfected with CV-B4.

In previous studies it was shown that inoculation of Swiss albino mice with CV-B4 E2 resulted in the production of serum IgG capable of enhancing the CV-B4 E2 infection of murine spleen cells cultures. To investigate whether such an enhancing activity of serum can play a role in vivo, we decided to study the CV-B4 E2 infection in mice exposed to successive inoculations of virus. In Swiss albino mice infected with CV-B4 E2 at the age of 21 days, anti-CV-B4 E2 neutralizing and enhancing activities of their serum peaked after 55 d. In contrast, mice inoculated at the age of 55 d expressed much lower activities. Despite the neutralizing activity of serum, CV-B4 E2 inoculated a second time to 55 day-old animals spread into the host. At the age of 72 and 89 d the levels of viral RNA and infectious particles were higher in organs of animals exposed to 2 successive infections compared with animals infected once at the age of 21 d or 55 d. In animals with 2 successive inoculations of CV-B4 E2 there was a relationship between the anti-CV-B4 E2 enhancing activity of serum and the level of viral RNA in organs and an enhancement of pathology was observed as displayed by histological analysis of pancreas and hyperglycaemia. Altogether our data strongly suggest that an anti-CV-B4 E2 enhancing activity in the host can play a role in the outcome of a secondary infection with this virus.

Central nervous system infection following vertical transmission of Coxsackievirus B4 in mice.

Coxsackie B viruses (CV-B) are important pathogens associated with several central nervous system (CNS) disorders. CV-B are mainly transmitted by the faecal-oral route, but there is also evidence for vertical transmission. The outcome of in utero CV-B infections on offspring's CNS is poorly explored. The aim of this study was to investigate vertical transmission of CV-B to the CNS. For this purpose, pregnant Swiss albino mice were intraperitoneally inoculated with CV-B4 E2 at gestational days 10G or 17G. Different CNS compartments were collected and analyzed for virus infection and histopathological changes. Using plaque assays, we demonstrated CV-B4 E2 vertical transmission to offspring's CNS. Viral RNA persisted in the CNS up to 60 days after birth, as evidenced by a sensitive semi-nested(sn) reverse transcripton(RT)-PCR method. This was despite infectious particles becoming undetectable at later time points. Persistence was associated with inflammatory lesions, lymphocyte infiltration and viral dsRNA detected by immunohistochemistry. Offspring born to dams mock- or virus-infected at day 17G were challenged by the same virus at day 21 after birth (-+ and ++ groups, respectively). Sn-RT-PCR and histology results compared between both ++ and -+ groups, show that in utero infection did not enhance CNS infection during challenge of the offspring with the same virus.

How Does Thymus Infection by Coxsackievirus Contribute to the Pathogenesis of Type 1 Diabetes?

Through synthesis and presentation of neuroendocrine self-antigens by major histocompatibility complex proteins, thymic epithelial cells (TECs) play a crucial role in programing central immune self-tolerance to neuroendocrine functions. Insulin-like growth factor-2 (IGF-2) is the dominant gene/polypeptide of the insulin family that is expressed in TECs from different animal species and humans. Igf2 transcription is defective in the thymus of diabetes-prone bio-breeding rats, and tolerance to insulin is severely decreased in Igf2 (-/-) mice. For more than 15 years now, our group is investigating the hypothesis that, besides a pancreotropic action, infection by coxsackievirus B4 (CV-B4) could implicate the thymus as well, and interfere with the intrathymic programing of central tolerance to the insulin family and secondarily to insulin-secreting islet ß cells. In this perspective, we have demonstrated that a productive infection of the thymus occurs after oral CV-B4 inoculation of mice. Moreover, our most recent data have demonstrated that CV-B4 infection of a murine medullary (m) TEC line induces a significant decrease in Igf2 expression and IGF-2 production. In these conditions, Igf1 expression was much less affected by CV-B4 infection, while Ins2 transcription was not detected in this cell line. Through the inhibition of Igf2 expression in TECs, CV-B4 infection could lead to a breakdown of central immune tolerance to the insulin family and promote an autoimmune response against insulin-secreting islet ß cells. Our major research objective now is to understand the molecular mechanisms by which CV-B4 infection of TECs leads to a major decrease in Igf2 expression in these cells.