Lucie Lamontagne

Crosstalk between intestinal microbiota, adipose tissue and skeletal muscle as an early event in systemic low-grade inflammation and the development of obesity and diabetes

Obesity is associated with a systemic chronic low‐grade inflammation that contributes to the development of metabolic disorders such as cardiovascular diseases and type 2 diabetes. However, the etiology of this obesity‐related pro‐inflammatory process remains unclear. Most studies have focused on adipose tissue dysfunctions and/or insulin resistance in skeletal muscle cells as well as changes in adipokine profile and macrophage recruitment as potential sources of inflammation. However, low‐grade systemic inflammation probably involves a complex network of signals interconnecting several organs. Recent evidences have suggested that disturbances in the composition of the gut microbial flora and alterations in levels of gut peptides following the ingestion of a high‐fat diet may be a cause of low‐grade systemic inflammation that may even precede and predispose to obesity, metabolic disorders or type 2 diabetes. This hypothesis is appealing because the gastrointestinal system is first exposed to nutrients and may thereby represent the first link in the chain of events leading to the development of obesity‐associated systemic inflammation. Therefore, the present review will summarize the latest advances interconnecting intestinal mucosal bacteria‐mediated inflammation, adipose tissue and skeletal muscle in a coordinated circuitry favouring the onset of a high‐fat diet‐related systemic low‐grade inflammation preceding obesity and predisposing to metabolic disorders and/or type 2 diabetes. A particular emphasis will be given to high‐fat diet‐induced alterations of gut homeostasis as an early initiator event of mucosal inflammation and adverse consequences contributing to the promotion of extended systemic inflammation, especially in adipose and muscular tissues. Copyright

Porcine reproductive and respiratory syndrome virus persistence in blood, spleen, lymph nodes, and tonsils of experimentally infected pigs depends on the level of CD8high T cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection suggesting an inefficient cellular immune response. The aim of the study was to evaluate the relationship between viral persistence and cytotoxic cells in blood, spleen, mediastinal lymph nodes (MLN) and tonsils of PRRSV experimentally infected pigs. Groups of four to six specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate, and blood and lymphoid organs were collected from 3 to 60 days post-infection (p.i.). Infectious particles and viral RNA were more or less rapidly eliminated in serum, spleen, lungs and MLN but persisted the longest in tonsils. Lymphocytes CD2+ CD4+, CD2+ CD8high, CD2+ CD8low and NK cells populations were phenotyped and their reactivity to PHA and ConA were tested. Analysis of T cell subsets in blood and lymphoid organs indicated that the percentages of CD2+ CD8+ T cells slightly increased in spleen at 17 days p.i, whereas no changes were observed in CD2+ CD4+ cells in blood or lymphoid organs. However, discrimination of CD8+ cells in CD8high and CD8low subsets revealed that the percentages of CD2+ CD8high cells increased in spleen and blood from 10 to 45 or 60 days p.i. while they transiently increased in MLN and decreased in tonsils. The CD8low/CD8high ratio increased in the blood of PRRSV-infected animals at three days p.i. due to a transient decrease of CD2+ CD8high cells. This same ratio decreased in the spleen of infected pigs from 10 to 45 days p.i. due to an increase of CD2+ CD8high cells. The CD2+ MIL-4+ cell subset (NK cells) was not significantly modified in blood or lymphoid organs. In addition, the ability of lymphoid T cells from blood and lymphoid organs to respond to ConA or PHA stimulation was transiently impaired in blood and spleen during the PRRSV persistent infection. Taken together, these results suggest that, in persistently infected pigs, an impaired CD2+ CD8high cell response in MLN and tonsils favors viral persistence in these organs, in contrast with the response seen in blood and spleen where viral elimination appears to occur sooner.

Polyclonal activation of B cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (PRRSV)-infected pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection associated with an inefficient humoral immune response. A study of lymphoid B cells and specific humoral immune response was performed in blood and several lymphoid organs collected from PRRSV experimentally-infected pigs. Groups of specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate of PRRSV, and blood, tonsils, spleen and mediastinal lymph nodes (MLN) were collected at various times postinfection (p.i.) (3-60 days). Lymphoid cells were isolated, immunolabeled for cytofluorometric determination of B cell percentages, used for counting specific anti-PRRSV antibody secreting B cells by an ELISPOT assay, or cultured for metabolic activity. The presence of anti-PRRSV antibodies in the serum of infected pigs was determined using a commercial ELISA assay. Virus detection was performed in all tissues, including lungs, by virus isolation and RT-PCR. The results show that percentages of B cells increased in tonsils as soon as 3 days until 17 days p.i. in PRRSV-infected pigs while they increased in spleen at 3 days p.i. only, due to an increase of larger Ig(high)-producing B cells. Metabolic activity of lymphoid cells from blood and spleen increased at 3 days p.i. only while lymphoid cells from tonsils and MLN transiently decreased at that time and increased thereafter up to 60 days p.i. Anti-PRRSV antibody-secreting B cells occurred in tonsils after 10 days p.i. and strongly increased up to 60 days p.i. However, specific anti-PRRSV-secreting B cells were detected in blood and spleen after 17 days p.i and in MLN only after 45 days p.i. Specific antibodies were detectable in serum at 10 days p.i., reached the maximum level at 45 days and remained high up to 60 days p.i. Infectious virus was detected in lungs and MLN as soon as 3 days p.i., and remained detectable up to 45 days p.i. in tonsils of one pig while viral RNA was detected in most organs up to 60 days p.i. In vitro experiments revealed that inactivated virus induced a stimulation of lymphoid cells isolated from PRRSV-infected pigs while it was cytotoxic for lymphoid cells from control pigs. Taken together, these results indicate that viral infection induced simultaneously a polyclonal activation of B cells, mainly in tonsils, and an exaggerated and prolonged specific humoral immune response due to persistent viral infection in lymphoid organs.

New Lactobacillus acidophilus isolates reduce the release of leptin by murine adipocytes leading to lower interferon-γ production

Leptin produced by adipocytes increases Th1‐dependent immunostimulation and autoimmune diseases. Lactobacilli are known to promote or suppress Th1 responses according to the isolates. We have investigated whether the sensitivity of Suriss Jim Lambert (SJL) mice to Th1‐dependent immune diseases, when compared with C57BL/6 mice, may be modulated by selected lactobacilli able to decrease leptin release by adipocytes. White adipocytes were isolated from both C57BL/6 and SJL mice and incubated with bacterial extracts from new CBA4P and TPA3P isolates of Lactobacillus acidophilus and L. rhamnosus 9595 (LR), or with conditioned media (CM) from lactobacillus‐treated macrophages. Immunomodulation induced by supernatants of treated adipocytes was determined by metabolic activity of syngenic splenic lymphocytes. Leptin produced by adipocytes, tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β by macrophages, and IFN‐γ and IL‐4 by lymphocytes were quantified by enzyme‐linked immunosorbent assay (ELISA) tests. Results revealed that supernatants from CBA4P‐ and LR‐treated adipocytes decreased the metabolic activity of lymphocytes from SJL mice, whereas adipocytes incubated with CM from CBA4P‐treated macrophages showed no stimulation of lymphocytes. Such effects correlated with leptin levels. Lower levels of leptin were produced by adipocytes from SJL mice in the presence of CBA4P and LR extracts. Lymphocytes from SJL mice produced low levels of IFN‐γ when incubated with supernatants from CBA4P‐treated cells. Such immunosuppressive effects were dependent on levels of TNF‐α and IL‐1β produced by lactobacillus‐treated macrophages. Taken together, these results suggest that the CBA4P isolate reduces levels of leptin in SJL mice, leading to lower IFN‐γ production. Therefore, the CBA4P isolate of L. acidophilus is a promising new probiotic strain for the control of Th1 inflammatory diseases.

Murine viral hepatitis involves NK cell depletion associated with virus‐induced apoptosis

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of immunological disorders related to acute and chronic hepatitis. In this study, we have verified if the fulminant hepatitis induced by MHV3 could be related to an impairment of innate immunity. Groups of three C57BL/6 mice were infected with the pathogenic L2‐MHV3 or attenuated YAC‐MHV3 viruses, and the natural killer (NK) cell populations from liver, spleen and bone marrow were analysed. The percentage of intrahepatic NK1·1+T cell receptor (TCR)− cells did not increase while NK1·1+TCRinter cells decreased in both L2‐MHV3‐ and YAC‐MHV3‐infected mice. Concurrently, splenic and myeloid NK1·1+ cells decreased in L2‐MHV3‐infected mice. However, the cytotoxic activity of NK cells increased in liver and decreased in bone marrow from pathogenic L2‐MHV3‐infected mice while no modification was detected in YAC‐MHV3‐infected mice. Flow cytometric analysis revealed that both normal and larger splenic or myeloid NK cells decreased more in pathogenic L2‐MHV3‐infected mice than in attenuated YAC‐MHV3‐infected mice. In vitro viral infections of interleukin (IL)‐15‐stimulated lymphoid cells from liver and bone marrow revealed that L2‐MHV3 induced higher decreases in cell viability of NK1·1+ cells than the YAC‐MHV3 variant. The NK cell decreases were due to the viral permissivity leading to cytopathic effects characterized by cell rounding, syncytia formation and apoptosis. Larger NK+ syncytia were observed in L2‐MHV3‐infected cells than in YAC‐MHV3‐infected cells. These results suggest that NK cell production is impaired by viral infection favouring fulminant hepatitis.

Macrophage interleukin‐6 and tumour necrosis factor‐α are induced by coronavirus fixation to Toll‐like receptor 2/heparan sulphate receptors but not carcinoembryonic cell adhesion antigen 1a

A rapid antiviral immune response may be related to viral interaction with the host cell leading to activation of macrophages via pattern recognition receptors (PPRs) or specific viral receptors. Carcinoembryonic cell adhesion antigen 1a (CEACAM1a) is the specific receptor for the mouse hepatitis virus (MHV), a coronavirus known to induce acute viral hepatitis in mice. The objective of this study was to understand the mechanisms responsible for the secretion of high‐pathogenic MHV3‐induced inflammatory cytokines. We report that the induction of the pro‐inflammatory cytokines interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α in peritoneal macrophages does not depend on CEACAM1a, as demonstrated in cells isolated from Ceacam1a−/− mice. The induction of IL‐6 and TNF‐α production was related rather to the fixation of the spike (S) protein of MHV3 on Toll‐like receptor 2 (TLR2) in regions enriched in heparan sulphate and did not rely on viral replication, as demonstrated with denatured S protein and UV‐inactivated virus. High levels of IL‐6 and TNF‐α were produced in livers from infected C57BL/6 mice but not in livers from Tlr2−/− mice. The histopathological observations were correlated with the levels of those inflammatory cytokines. Depending on mouse strain, the viral fixation to heparan sulfate/TLR2 stimulated differently the p38 mitogen‐activated protein kinase (MAPK) and nuclear factor (NF)‐κB in the induction of IL‐6 and TNF‐α. These results suggest that TLR2 and heparan sulphate receptors can act as new viral PPRs involved in inflammatory responses.

Pathogenic Mouse Hepatitis Virus or Poly(I:C) Induce IL-33 in Hepatocytes in Murine Models of Hepatitis

The IL-33/ST2 axis is known to be involved in liver pathologies. Although, the IL-33 levels increased in sera of viral hepatitis patients in human, the cellular sources of IL-33 in viral hepatitis remained obscure. Therefore, we aimed to investigate the expression of IL-33 in murine fulminant hepatitis induced by a Toll like receptor (TLR3) viral mimetic, poly(I:C) or by pathogenic mouse hepatitis virus (L2-MHV3). The administration of poly(I:C) plus D-galactosamine (D-GalN) in mice led to acute liver injury associated with the induction of IL-33 expression in liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells (VEC), while the administration of poly(I:C) alone led to hepatocyte specific IL-33 expression in addition to vascular IL-33 expression. The hepatocyte-specific IL-33 expression was down-regulated in NK-depleted poly(I:C) treated mice suggesting a partial regulation of IL-33 by NK cells. The CD1d KO (NKT deficient) mice showed hepatoprotection against poly(I:C)-induced hepatitis in association with increased number of IL-33 expressing hepatocytes in CD1d KO mice than WT controls. These results suggest that hepatocyte-specific IL-33 expression in poly(I:C) induced liver injury was partially dependent of NK cells and with limited role of NKT cells. In parallel, the L2-MHV3 infection in mice induced fulminant hepatitis associated with up-regulated IL-33 expression as well as pro-inflammatory cytokine microenvironment in liver. The LSEC and VEC expressed inducible expression of IL-33 following L2-MHV3 infection but the hepatocyte-specific IL-33 expression was only evident between 24 to 32h of post infection. In conclusion, the alarmin cytokine IL-33 was over-expressed during fulminant hepatitis in mice with LSEC, VEC and hepatocytes as potential sources of IL-33.

Intrahepatic endothelial and Kupffer cells involved in immunosuppressive cytokines and natural killer (NK)/NK T cell disorders in viral acute hepatitis

During acute viral hepatitis, the intrahepatic tolerance sustained by immunosuppressive cytokines such as interleukin (IL)‐4, IL‐10, transforming growth factor (TGF)‐β and prostaglandin E2 (PGE2), produced by Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), natural killer (NK) T cells and natural regulatory T cells may be disturbed. NK cells are recruited normally in the liver and produce interferon (IFN)‐γ to control viral replication. The use of mouse hepatitis virus type 3 (MHV3) attenuated variants showing selected tropisms for KC or LSEC have allowed determining their roles in the disturbances of immune tolerance during viral hepatitis. Groups of C57BL/6 mice were infected with the pathogenic L2‐MHV3 (KC+, LSEC+), low attenuated 51·6‐MHV3 (KC+, LSEC‐) or high attenuated CL12‐MHV3 (KC‐, LSEC‐) variants for the first 3 days. Results showed that IL‐10, TGF‐β and PGE2 production in the liver decreased in L2‐MHV3‐infected mice and increased in 51·6‐MHV3‐ and CL12‐MHV3‐infected mice. The ratio of IFN‐γ/IL‐4 in liver decreased in L2‐MHV3‐infected mice, while it was not (or low) altered in mice infected with the attenuated MHV3 variant mice. Phenotypic analysis of intrahepatic mononuclear cells revealed that apoptotic NK and NK T cells increased in mice infected with the L2‐MHV3, but were minor in 51·6‐MHV3‐ and CL12‐MHV3‐infected mice. The numbers of CD4+ forkhead box P3+ cells increased in the livers from low pathogenic CL12‐MHV3 and YAC‐MHV3‐infected mice. These results indicate that viral permissivity of KC and LSEC is involved in the decrease of IL‐10 and PGE2, while KC may play an additional role in the apoptosis of NK and NK T cells during acute viral hepatitis.

Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells

ABSTRACT Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. IMPORTANCE Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion.

Dose-dependent apoptosis induced by low concentrations of methylmercury in murine splenic Fas+ T cell subsets.

Methylmercury chloride (MeHgCl) is known to induce cellular and humoral immunodeficiencies in mice. In this study, the involvement of lymphoid subset disorders due to low concentrations of methylmercury (0.001-1.0 microM) has been examined. Cytofluorometric analysis of splenic cells exposed in vitro to low concentrations of MeHgCl for 48 h revealed two distinct populations: the first expressed a typical lymphoid forward light scatter (FSC)/side light scatter (SSC) pattern (R1 region), and the second was characterized by a lower FSC and a higher SSC (R2 region). A dose-dependent shift of cells from R1 region toward R2 region was observed in splenic cells treated with MeHgCl. The apoptotic state of cells in the R2 region was confirmed by the TdT-mediated dUTP nick end labeling (TUNEL) assay. Analysis of DNA content in splenic lymphoid cells showed that low concentrations of MeHgCl increased both hypoploid cells and cells in G0-G1/S phase, both in the R1 and R2 regions. However, the numbers of cells in G0-G1/S and G2/M phases were decreased, but hypoploid cells increased in both regions due to exposure to 1 microM MeHgCl. MeHgCl-induced apoptosis disappeared when splenic cells were pretreated with anti-Fas antibodies, indicating that Fas expressing cells were the target cells for MeHgCl-mediated apoptosis. Furthermore, T cells from the V beta 8 family were found to be more sensitive to apoptosis induced by low concentrations of MeHgCl. Taken together, these results suggest that MeHgCl at low concentrations mediates the development of apoptosis in peripheral T cell via the Fas/FasL pathway.