Macrophages Are Targeted by Rotavirus in Experimental Biliary Atresia and Induce Neutrophil Chemotaxis by Mip2/Cxcl2

Abstract

Biliary atresia is an obstructive cholangiopathy of unknown etiology. Although the adaptive immune system has been shown to regulate the obstruction of bile ducts in a rotavirus-induced mouse model, little is known about the virus-induced inflammatory response. Here, we hypothesized that cholangiocytes secrete chemoattractants in response to rotavirus. To test this hypothesis, we infected cholangiocyte and macrophage cell lines with rhesus rotavirus type A (RRV), quantified cytokines and chemokines and measured the migration of splenocytes. We also used PCR and immunostaining to search for new cellular targets of RRV in the liver. We found that RRV-infected cholangiocytes induced the mRNA expression for chemokines, but conditioned media failed to promote chemotaxis of splenocytes. Analyzing livers after viral challenge, we detected RRV in hepatic macrophages and demonstrated that media from RRV-infected macrophages have high concentrations of cytokines and chemokines and induced chemotaxis of neutrophils. Most notably, addition of anti-Mip2/Cxcl2 antibodies depleted this chemokine in the conditioned media and completely prevented neutrophil chemotaxis. In conclusion, infected cholangiocytes did not promote chemotaxis of inflammatory cells. Investigating alternate cellular targets of RRV, we detected the virus in hepatic macrophages and found that infected macrophages promoted neutrophil chemotaxis by release of Mip2/Cxcl2 in response to RRV. (Pediatr Res 67: 345-351, 2010) B iliary atresia, the most common cause of neonatal cholestasis, results from an inflammatory and fibrosing obstruction of extrahepatic bile ducts. The etiology is unknown, but studies in an experimental mouse model of rotavirus-induced biliary atresia indicate that pathogenic mechanisms of disease begin with an injury to the biliary epithelium, followed by a robust inflammatory infiltration of the wall of extrahepatic bile ducts, obstruction of the lumen by inflammatory cells, and final progression to fibrosis (1). These pathologic changes in the liver and extrahepatic bile ducts closely resemble human biliary atresia (2,3). Studies of livers of infants at the time of diagnosis have long recognized an activation of inflammatory cells (4), and a broad analysis of the hepatic gene expression profile identified a prominent proinflammatory signature (5). Disruption of this signature by loss of IFN-␥ or the loss of CD8ϩ lymphocytes in the mouse model largely prevented duct obstruction and the phenotype of experimental biliary atresia (6,7). Interestingly, in vivo depletion of CD4ϩ lymphocytes or the genetic loss of IL-12, or the depletion of TNF-␣ later in the course of biliary injury did not alter the progression to biliary atresia phenotype (7-9), which supported the coexistence of accessory pathways regulating the pathology of extrahepatic bile ducts. Despite the progress in deciphering key elements regulating duct obstruction and atresia, little is known about molecular circuits that are activated in early stages of the disease.Cellu...

Dendritic Cells Regulate Natural Killer Cell Activation and Epithelial Injury in Experimental Biliary Atresia

“…Here, we provide evidence for an alternate possibility that hepatic DCs are cellular targets of RRV and induce proliferation and activation of CD8 + T cells and NK cells, both of which have been shown to injure infected cholangiocytes and damage the duct epithelium in vivo. These results do not exclude a potential participation of cholangiocytes in attracting lymphocytes or myeloid cells after RRV infection, but experiments to quantify this response revealed relatively low levels of cytokines and chemokines in RRV-infected cholangiocytes (25, 26). Our data also do not exclude a potential role for cholangiocytes in the promotion of T cell proliferation in vivo when aided by other cells (for example, NK cells) in response to RRV infection.…”

“…DCs are uniquely fitted to sense pathogens and to orchestrate an immune response by establishing intercellular circuits among themselves and with other cells (20, 26, 27). In adult mice, cDCs are the major source of IL-15 production in response to CpG oligonucleotides but require accessory signals from pDCs for appropriate responses, perhaps mediated by CD40L (20).…”

Identification of a plant isoflavonoid that causes biliary atresia

“…3), suggesting a potential contributory role for the innate immune system in toxin-induced biliary injury as identified in the RRV BA model (19). Late accumulation of macrophages around injured intrahepatic ducts is also seen in RRV-infected mice (11), where they are thought to attract neutrophils (32). Macrophage depletion also prevented duct obstruction in the RRV model (33).…”

Comparative In Vitro and In Vivo Studies of Porcine Rotavirus G9P[13] and Human Rotavirus Wa G1P[8]

“…Others have suggested that in rhesus macaques, rhesus RV (RRV) escapes the intestine via a lymphatic route, with the viral nonstructural proteins (NSPs) being sequentially detected in Peyer's patches, mesenteric lymph nodes (MLNs), spleen, and liver (33). RV NSPs have been observed in mouse macrophages as well as human macrophages, dendritic cells (DCs), and B cells (34)(35)(36). These results suggest that immune cells may be permissive to RV infection and may play a role in RV persistence and extraintestinal dissemination.…”