Christian Bleau

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

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.

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.

Chicken infectious anaemia vaccinal strain persists in the spleen and thymus of young chicks and induces thymic lymphoid cell disorders

The chicken infectious anaemia virus (CIAV) infection may induce immunosuppression and persistent infection. The use of vaccination in young chicks is still controversial due to its low immune efficiency. In order to verify the viral persistency of a vaccinal strain of CIAV and its associated-lymphoid cell disorders, 54 1-day-old specific pathogen free chicks were vaccinated (CIAV-VAC®; Intervet, Millsboro, Delaware, USA) and haematologic examination, expression of viral VP3 gene, humoral response and phenotyping of lymphoid cells were studied in lymphoid organs at various times post vaccination (p.v.). No clinical signs were observed but light heteropaenia was detected in CIAV-vaccinated chicks. The VP3 gene of CIAV was detected by polymerase chain reaction in the thymus and spleen from day 7 until 28 days p.v. Thymic larger CD4+CD8+ cells increased only at 7 days p.v. while smaller CD4+CD8+ cells decreased after 14 and 28 days in CIAV-vaccinated birds. The CD4 expression, in contrast to that seen for CD8, decreased in thymocytes from the CIAV-vaccinated group. In the spleen and bursa, the percentage of CD8+ cells increased at 7 and 28 days p.v. only, while CD4+ cells decreased simultaneously. The vaccinated chicks also exhibited a higher number of splenic CD3–CD8+ cells (natural killer cells). The anti-CIAV antibody responses, however, remained low in most vaccinated chicks and did not persist up to 18 days p.v. These results suggest that the vaccinal virus strain is clinically attenuated but persists in the thymus and spleen in some birds, inducing a low humoral immune response and altering thymopoiesis.

Mouse Hepatitis Virus Infection Induces a Toll-Like Receptor 2-Dependent Activation of Inflammatory Functions in Liver Sinusoidal Endothelial Cells during Acute Hepatitis

ABSTRACT Under physiological conditions, the liver sinusoidal endothelial cells (LSECs) mediate hepatic immune tolerance toward self or foreign antigens through constitutive expression of anti-inflammatory mediators. However, upon viral infection or Toll-like receptor 2 (TLR2) activation, LSECs can achieve proinflammatory functions, but their role in hepatic inflammation during acute viral hepatitis is unknown. Using the highly virulent mouse hepatitis virus type 3 (MHV3) and the attenuated variants 51.6-MHV3 and YAC-MHV3, exhibiting lower tropism for LSECs, we investigated in vivo and in vitro the consequence of LSEC infection on their proinflammatory profiles and the aggravation of acute hepatitis process. In vivo infection with virulent MHV3, in comparison to attenuated strains, resulted in fulminant hepatitis associated with higher hepatic viral load, tissue necrosis, and levels of inflammatory mediators and earlier recruitment of inflammatory cells. Such hepatic inflammatory disorders correlated with disturbed production of interleukin-10 (IL-10) and vascular factors by LSECs. We next showed in vitro that infection of LSECs by the virulent MHV3 strain altered their production of anti-inflammatory cytokines and promoted higher release of proinflammatory and procoagulant factors and earlier cell damage than infection by attenuated strains. This higher replication and proinflammatory activation in LSECs by the virulent MHV3 strain was associated with a specific activation of TLR2 signaling by the virus. We provide evidence that TLR2 activation of LSCEs by MHV3 is an aggravating factor of hepatic inflammation and correlates with the severity of hepatitis. Taken together, these results indicate that preservation of the immunotolerant properties of LSECs during acute viral hepatitis is imperative in order to limit hepatic inflammation and damage. IMPORTANCE Viral hepatitis B and C infections are serious health problems affecting over 350 million and 170 million people worldwide, respectively. It has been suggested that a balance between protection and liver damage mediated by the hosts immune response during the acute phase of infection would be determinant in hepatitis outcome. Thus, it appears crucial to identify the factors that predispose in exacerbating liver inflammation to limit hepatocyte injury. Liver sinusoidal endothelial cells (LSECs) can express both anti- and proinflammatory functions, but their role in acute viral hepatitis has never been investigated. Using mouse hepatitis virus (MHV) infections as animal models of viral hepatitis, we report for the first time that in vitro and in vivo infection of LSECs by the pathogenic MHV3 serotype leads to a reversion of their intrinsic anti-inflammatory phenotype toward a proinflammatory profile as well to as disorders in vascular factors, correlating with the severity of hepatitis. These results highlight a new virus-promoted mechanism of exacerbation of liver inflammatory response during acute hepatitis.

Toll‐like receptor (tlr)‐2 exacerbates murine acute viral hepatitis

Viral replication in the liver is generally detected by cellular endosomal Toll‐like receptors (TLRs) and cytosolic helicase sensors that trigger antiviral inflammatory responses. Recent evidence suggests that surface TLR2 may also contribute to viral detection through recognition of viral coat proteins but its role in the outcome of acute viral infection remains elusive. In this study, we examined in vivo the role of TLR2 in acute infections induced by the highly hepatotrophic mouse hepatitis virus (MHV) type 3 and weakly hepatotrophic MHV‐A59 serotype. To address this, C57BL/6 (wild‐type; WT) and TLR2 knockout (KO) groups of mice were intraperitoneally infected with MHV3 or MHV‐A59. MHV3 infection provoked a fulminant hepatitis in WT mice, characterized by early mortality and high alanine and aspartate transaminase levels, histopathological lesions and viral replication whereas infection of TLR2 KO mice was markedly less severe. MHV‐A59 provoked a comparable mild and subclinical hepatitis in WT and TLR2 KO mice. MHV3‐induced fulminant hepatitis in WT mice correlated with higher hepatic expression of interferon‐β, interleukin‐6, tumour necrosis factor‐α, CXCL1, CCL2, CXCL10 and alarmin (interleukin‐33) than in MHV‐A59‐infected WT mice and in MHV3‐infected TLR2 KO mice. Intrahepatic recruited neutrophils, natural killer cells, natural killer T cells or macrophages rapidly decreased in MHV3‐infected WT mice whereas they were sustained in MHV‐A59‐infected WT mice and MHV3‐infected TLR2 KO. MHV3 in vitro infection of macrophagic cells induced rapid and higher viral replication and/or interleukin‐6 induction in comparison to MHV‐A59, and depended on viral activation of TLR2 and p38 mitogen‐activated protein kinase. Taken together, these results support a new aggravating inflammatory role for TLR2 in MHV3‐induced acute fulminant hepatitis.

A synergistic interferon-γ production is induced by mouse hepatitis virus in interleukin-12 (IL-12)/IL-18-activated natural killer cells and modulated by carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1a receptor

The production of interferon‐γ (IFN‐γ) by infiltrating natural killer (NK) cells in liver is involved in the control of mouse hepatitis virus (MHV) infection. The objectives of this study were to identify the mechanisms used by MHV type 3 to modulate the production of IFN‐γ by NK cells during the acute hepatitis in susceptible C57BL/6 mice. Ex vivo and in vitro experiments revealed that NK cells, expressing carcinoembryonic antigen‐related cell adhesion molecules (CEACAM) 1a (the MHV receptor), can produce a higher level of IFN‐γ in the presence of both L2‐MHV3 and interleukin‐12 (IL‐12)/IL‐18. The synergistic production of IFN‐γ by NK cells depends on viral replication rather than viral fixation only, because it is inhibited or not induced in cells infected with ultraviolet‐inactivated viruses and in cells from Ceacam1a−/− mice infected with virulent viruses. The synergistic IFN‐γ production involves the p38 mitogen‐activated protein kinase (MAPK) rather than the extracellular signal‐regulated kinase‐1/2 MAPK signalling pathway. However, the signal triggered through the engagement of CEACAM1a decreases the production of IFN‐γ, when these molecules are cross‐linked using specific monoclonal antibodies. These results suggest that control of acute hepatitis by IFN‐γ‐producing NK cells may depend on both production of IL‐12 and IL‐18 in the liver environment and viral infection of NK cells.