BACKGROUND & AIMS Capillarization, characterized by loss of differentiation of liver sinusoidal endothelial cell (LSEC), precedes the onset of hepatic fibrosis. We investigated whether restoring differentiation to LSEC in liver affects their interactions with hepatic stellate cells (HSCs) and thereby promotes quiescence of HSCs and regression of fibrosis. METHODS Rat LSECs were cultured with inhibitors and/or agonists and examined by scanning electron microscopy for fenestrae in sieve plates. Cirrhosis was induced in rats using thioacetamide, followed by administration of BAY 60-2770, an activator of soluble guanylate cyclase (sGC). Fibrosis was assessed by Sirius red staining; expression of α-smooth muscle actin was measured by immunoblot analysis. RESULTS Maintenance of LSEC differentiation requires vascular endothelial growth factor-A stimulation of nitric oxide (NO)-dependent signaling (via sGC and cGMP) and NO-independent signaling. In rats with thioacetamide-induced cirrhosis, BAY 60-2770 accelerated the complete reversal of capillarization (restored differentiation of LSEC) without directly affecting activation of HSC or fibrosis. Restoration of differentiation to LSEC led to quiescence of HSC and regression of fibrosis, in the absence of further exposure to BAY 60-2770. Activation of sGC with BAY 60-2770, prevented progression of cirrhosis, despite continued administration of thioacetamide. CONCLUSIONS Differentiation of LSEC has an important role in activation of HSC and the fibrotic process in rats.

Contributions of humoral and cellular immunity in controlling neurotropic mouse hepatitis virus persistence within the CNS were determined in B cell-deficient JHD and syngeneic H-2d B cell+ Ab-deficient mice. Virus clearance followed similar kinetics in all mice, confirming initial control of virus replication by cellular immunity. Nevertheless, virus reemerged within the CNS of all Ab-deficient mice. In contrast to diminished T cell responses in H-2b B cell-deficient μMT mice, the absence of B cells or Ab in the H-2d mice did not compromise expansion, recruitment into the CNS, or function of virus-specific CD4+ and CD8+ T cells. The lack of B cells and lymphoid architecture thus appears to manifest itself on T cell responses in a genetically biased manner. Increasing viral load did not enhance frequencies or effector function of virus-specific T cells within the CNS, indicating down-regulation of T cell responses. Although an Ab-independent antiviral function of B cells was not evident during acute infection, the presence of B cells altered CNS cellular tropism during viral recrudescence. Reemerging virus localized almost exclusively to oligodendroglia in B cell+ Ab-deficient mice, whereas it also replicated in astrocytes in B cell-deficient mice. Altered tropism coincided with distinct regulation of CNS virus-specific CD4+ T cells. These data conclusively demonstrate that the Ab component of humoral immunity is critical in preventing virus reactivation within CNS glial cells. B cells themselves may also play a subtle role in modulating pathogenesis by influencing tropism.

Neurotropic coronavirus infection induces expression of both beta interferon (IFN-␤

Neurotropic coronavirus infection of mice results in acute encephalomyelitis followed by viral persistence. Whereas cellular immunity controls acute infection, humoral immunity regulates central nervous system (CNS) persistence. Maintenance of serum Ab was correlated with tissue distribution of virus-specific Ab-secreting cells (ASC). Although virus-specific ASC declined in cervical lymph node and spleen after infectious virus clearance, virus-specific serum Ab was sustained at steady levels, with a delay in neutralizing Ab. Virus-specific ASC within the CNS peaked rapidly 1 wk after control of infectious virus and were retained throughout chronic infection, consistent with intrathecal Ab synthesis. Surprisingly, frequencies of ASC in the BM remained low and only increased gradually. Nevertheless, virus-specific ASC induced by peripheral infection localized to both spleen and BM. The data suggest that CNS infection provides strong stimuli to recruit ASC into the inflamed tissue through sustained up-regulation of the CXCR3 ligands CXCL9 and CXCL10. Irrespective of Ag deprivation, CNS retention of ASC coincided with elevated BAFF expression and ongoing differentiation of class II + to class II -CD138 + CD19 + plasmablasts. These results confirm the CNS as a major ASCsupporting environment, even after resolution of viral infection and in the absence of chronic ongoing inflammation. IntroductionThe preeminent goal of the immune system is to eliminate pathogens and establish immunological memory [1]. Both T cells and Ab participate in eliminating a variety of pathogens; however, sustained serum Ab is an important criteria for many vaccination strategies, as they provide the first line of defense against re-infection [2]. Upon Ag encounter in regional lymph nodes, B cells undergo clonal expansion in extrafollicular foci and within germinal centers [3,4].Rapidly activated B cells secrete low-affinity Ab but can undergo isotype switching and limited BCR hypermutation as they differentiate into plasmablasts [3]. In the milieu of accessory cells and cytokines, germinal center B cells undergo affinity maturation and ultimately differentiate into both Ab-secreting cells (ASC) and memory B cells. As Ag is depleted, ASC and memory B cells are detected with increasing frequency in BM [2], where both stromal cells and other resident cells provide soluble as well as contact-dependent survival signals, including CXCL12 and BAFF [5]. Ab secretion by terminally differentiated plasma cells is independent of both Ag and T cell regulation [2,6]. Long-lived ASC in BM and spleen maintain serum Ab, thus providing protective immunity to re-infection, sometimes for the [4,6]. In contrast to Ag encountered in the periphery, the regulation of B cell activation by Ag sequestered within the central nervous system (CNS) is less clear. The absence of dedicated lymphatic drainage and the presence of the blood brain barrier (BBB) limits Ag transport from the CNS into secondary lymphoid tissue as well as trafficking of both cells and macromolecule...

Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.

Infection of the central nervous system (CNS) by the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces an acute encephalomyelitis associated with demyelination. To examine the anti-viral and/or regulatory role of interferon-gamma (IFN-gamma) signaling in the cell that synthesizes and maintains the myelin sheath, we analyzed JHMV pathogenesis in transgenic mice expressing a dominant-negative IFN-gamma receptor on oligodendroglia. Defective IFN-gamma signaling was associated with enhanced oligodendroglial tropism and delayed virus clearance. However, the CNS inflammatory cell composition and CD8(+) T-cell effector functions were similar between transgenic and wild-type mice, supporting unimpaired peripheral and CNS immune responses in transgenic mice. Surprisingly, increased viral load in oligodendroglia did not affect the extent of myelin loss, the frequency of oligodendroglial apoptosis, or CNS recruitment of macrophages. These data demonstrate that IFN-gamma receptor signaling is critical for the control of JHMV replication in oligodendroglia. In addition, the absence of a correlation between increased oligodendroglial infection and the extent of demyelination suggests a complex pathobiology of myelin loss in which infection of oligodendroglia is required but not sufficient.

The neurotropic coronavirus JHM strain of mouse hepatitis virus persists in oligodendroglia despite the presence of virusspecific CD8 T cells. Expression of programmed death 1 (PD-1) and B7-H1 were studied during acute and persistent infection to examine whether this negative regulatory mechanism contributes to CNS viral persistence. The majority of CNS-infiltrating CD8 T cells expressed PD-1, with the highest levels on virus-specific CD8 T cells. Moreover, despite control of infectious virus, CD8 T cells within the CNS of persistently infected mice maintained high PD-1 expression. Analysis of virus-susceptible target cells in vivo revealed that B7-H1 expression was regulated in a cell type-dependent manner. Oligodendroglia and microglia up-regulated B7-H1 following infection; however, although B7-H1 expression on oligodendroglia was prominent and sustained, it was significantly reduced and transient on microglia S everal features of the CNS make it a challenging environment for the immune response to efficiently clear virus. These obstacles include the blood-brain barrier, a specialization of the vasculature in the CNS that limits leukocyte entry (1). In addition, the absence of classical lymphoid drainage and restricted Ag presentation by CNS resident cells hinders induction, recruitment, and retention of adaptive immune cells. Furthermore, in contrast to peripheral infections, several target cells of CNS infections are terminally differentiated, nonrenewable cells which have evolved mechanisms to avoid extensive immune-mediated damage. However, the same mechanisms protecting CNS resident cells from extensive immune-mediated pathology also favor establishment of viral persistence.A variety of inhibitory molecules and their ligands on T cells and infected cells contribute to T cell dysfunction, thereby fostering viral persistence. Among these are the programmed death 1 (PD-1) 5 receptor and its ligand B7-H1, also known as PD-L1, which belong to the B7:CD28 family (2). PD-1 is inducibly expressed on T cells, B cells (3), monocytes (4), and NK cells (5). B7-H1 is constitutively expressed on T cells, B cells, macrophages, and dendritic cells and is further up-regulated after stimulation (6, 7). B7-H1 is also expressed on a variety of nonhematopoietic cell types including vascular endothelial cells (8), epithelial cells (9), and cells of the nervous system (10 -13). PD-1:B7-H1 interactions trigger a noneffective T cell response, referred to as "T cell exhaustion," which is characterized by impaired proliferation, cytolysis, and cytokine production (8, 9, 14 -24). Exhausted T cells were initially described in chronic lymphocytic choriomeningitis virus-infected mice (24) and similar dysfunction of T cells has been reported during HIV (19, hepatitis C virus (15,26,27)-, and simian immunodeficiency virus (28)-persistent infections. Similar to pathogens exploiting the PD-1:B7-H1 pathway to establish persistence, expression of B7-H1 on tumors may also serve as an immune evasion strategy (29,30).The role of inhibitory mol...

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-γ-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-γ ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.