Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity.

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxin- and model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses.

Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay.

Dengue is a major public health problem globally. It is caused by four antigenically distinct serotypes of dengue virus (DENV1-4), and although serotype-specific and strongly neutralizing cross-reactive immune responses against the four DENV serotypes are thought to be protective, subneutralizing Abs can contribute to increased disease severity upon secondary infection with a different DENV serotype. Understanding the breadth of the immune response in natural DENV infections and in vaccinees is crucial for determining the correlates of protection or disease severity. Transformation of B cell populations to generate mAbs and ELISPOT assays have been used to determine B cell and Ab specificity to DENV; however, both methods have technical limitations. We therefore modified the conventional ELISPOT to develop a Quad-Color FluoroSpot to provide a means of examining B cell/Ab serotype specificity and cross-reactivity on a single-cell basis. Abs secreted by B cells are captured by an Fc-specific Ab on a filter plate. Subsequently, standardized concentrations of all four DENV serotypes are added to allow equal stoichiometry for Ag binding. After washing, the spots, representing individual B cells, are visualized using four fluorescently labeled DENV serotype-specific detection mAbs. This method can be used to better understand the breadth and magnitude of B cell responses following primary and secondary DENV infection or vaccination and their role as immune correlates of protection from subsequent DENV infections. Furthermore, the Quad-Color FluoroSpot assay can be applied to other diseases caused by multiple pathogen serotypes in which determining the serotype or subtype-specific B cell response is important.

Antibody avidity following secondary dengue virus type 2 infection across a range of disease severity.

BACKGROUND: The four dengue virus serotypes (DENV1-4) are responsible for the most prevalent mosquito-borne viral illness in humans. DENV causes a spectrum of disease from self-limiting dengue fever (DF) to severe, life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Antibodies from one infection can contribute to either protection or increased disease severity in a subsequent infection with a distinct DENV serotype. The effectiveness of the antibody response is modulated by both the affinity and avidity of the antibody/antigen interaction. OBJECTIVES: We investigated how antibody avidity developed over time following secondary DENV2 infection across different disease severities. STUDY DESIGN: We analyzed sera from 42 secondary DENV2-infected subjects (DF, n=15; DHF, n=16; DSS, n=11) from a pediatric hospital-based dengue study in Nicaragua. IgG avidity against DENV2 virions was measured in samples collected during acute and convalescent phases as well as 3, 6, and 18 months post-illness using a urea enzyme-linked immunosorbent assay. RESULTS: The data show a significant increase in avidity from acute to convalescent phase followed by a decrease from convalescent phase to 3 months post-symptom onset, then a plateau. Linear regression analysis comparing antibody avidity between disease severity groups over time indicate that individuals with more severe disease (DHF/DSS) experienced greater decay in antibody avidity over time compared to less severe disease (DF), and ROC curve analysis showed that at 18 months post-illness, lower avidity was associated with previously having experienced more severe disease. CONCLUSIONS: These data suggest that increased dengue disease severity is associated with lower antibody avidity at later time-points post-illness.

Monocyte-plasmablast crosstalk during dengue.

Dengue virus infection induces a dramatic expansion of B cell plasmablasts. In this issue, Kwissa et al. (2014) begin with transcriptomic analysis and then integrate studies in human clinical samples, nonhuman primates, and coculture of primary human cells to identify a role for CD14(+)CD16(+) monocytes in generating plasmablast responses during dengue virus infection.

Innate immunity to dengue virus infection and subversion of antiviral responses.

Dengue is a major public health issue in tropical and subtropical regions worldwide. The four serotypes of dengue virus (DENV1-DENV4) are spread primarily by Aedes aegypti and Aedes albopictus mosquitoes, whose geographic range continues to expand. Humans are the only host for epidemic strains of DENV, and the virus has developed sophisticated mechanisms to evade human innate immune responses. The host cell's first line of defense begins with an intracellular signaling cascade resulting in production of interferon α/ß (IFN-α/ß), which promotes intracellular antiviral responses and helps initiates the adaptive response during the course of DENV infection. In response, DENV has developed numerous ways to subvert these intracellular antiviral responses and directly inhibit cellular signaling cascades. Specifically, DENV manipulates the unfolded protein response and autophagy to counter cellular stress and delay apoptosis. The DENV non-structural protein NS4B and subgenomic flavivirus RNA interfere with the RNA interference pathway by inhibiting the RNase Dicer. During heterotypic secondary DENV infection, subneutralizing antibodies can enable viral uptake through Fcγ receptors and down-regulate signaling cascades initiated via the pattern recognition receptors TLR-3 and MDA5/RIG-I, thus reducing the antiviral state of the cell. The DENV NS2B/3 protein cleaves human STING/MITA, interfering with induction of IFN-α/ß. Finally, DENV NS2A, NS4A, and NS4B complex together to block STAT1 phosphorylation, while NS5 binds and promotes degradation of human STAT2, thus preventing formation of the STAT1/STAT2 heterodimer and its transcriptional induction of interferon stimulating genes. Here, we discuss the host innate immune response to DENV and the mechanisms of immune evasion that DENV has developed to manipulate cellular antiviral responses.

IFN-γ from CD4 T cells is essential for host survival and enhances CD8 T cell function during Mycobacterium tuberculosis infection.

IFN-γ is necessary in both humans and mice for control of Mycobacterium tuberculosis. CD4 T cells are a significant source of IFN-γ during acute infection in mice and are required for control of bacterial growth and host survival. However, several other types of cells can and do produce IFN-γ during the course of the infection. We sought to determine whether IFN-γ from sources other than CD4 T cells was sufficient to control M. tuberculosis infection and whether CD4 T cells had a role in addition to IFN-γ production. To investigate the role of IFN-γ from CD4 T cells, a murine adoptive transfer model was developed in which all cells were capable of producing IFN-γ, with the exception of CD4 T cells. Our data in this system support that CD4 T cells are essential for control of infection, but also that IFN-γ from CD4 T cells is necessary for host survival and optimal long-term control of bacterial burden. In addition, IFN-γ from CD4 T cells was required for a robust CD8 T cell response. IFN-γ from T cells inhibited intracellular replication of M. tuberculosis in macrophages, suggesting IFN-γ may be necessary for intracellular bactericidal activity. Thus, although CD4 T cells play additional roles in the control of M. tuberculosis infection, IFN-γ is a major function by which these cells participate in resistance to tuberculosis.

CD4 T cell depletion exacerbates acute Mycobacterium tuberculosis while reactivation of latent infection is dependent on severity of tissue depletion in cynomolgus macaques.

CD4 T cells are believed to be important in protection against Mycobacterium tuberculosis, but the relative contribution to control of initial or latent infection is not known. Antibody-mediated depletion of CD4 T cells in M. tuberculosis-infected cynomolgus macaques was used to study the role of CD4 T cells during acute and latent infection. Anti-CD4 antibody severely reduced levels of CD4 T cells in blood, airways, and lymph nodes. Increased pathology and bacterial burden were observed in CD4-depleted monkeys during the first 8 weeks of infection compared to controls. CD4-depleted monkeys had greater interferon (IFN)-γ expression and altered expression of CD8 T cell activation markers. During latent infection, CD4 depletion resulted in clinical reactivation in only three of six monkeys. Reactivation was associated with lower CD4 T cells in the hilar lymph nodes. During both acute and latent infection, CD4 depletion was associated with reduced percentages of CXCR3(+) expressing CD8 T cells, reported to be involved in T cell recruitment, regulatory function, and effector and memory T cell maturation. CXCR3(+) CD8 T cells from hilar lymph nodes had more mycobacteria-specific cytokine expression and greater coexpression of multiple cytokines compared to CXCR3(-) CD8 T cells. CD4 T cells are required for protection against acute infection but reactivation from latent infection is dependent on the severity of depletion in the draining lymph nodes. CD4 depletion influences CD8 T cell function. This study has important implications for human HIV-M. tuberculosis coinfection.

The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes.

The transcription factors that regulate differentiation into the monocyte subset in bone marrow have not yet been identified. Here we found that the orphan nuclear receptor NR4A1 controlled the differentiation of Ly6C- monocytes. Ly6C- monocytes, which function in a surveillance role in circulation, were absent from Nr4a1-/- mice. Normal numbers of myeloid progenitor cells were present in Nr4a1-/- mice, which indicated that the defect occurred during later stages of monocyte development. The defect was cell intrinsic, as wild-type mice that received bone marrow from Nr4a1-/- mice developed fewer patrolling monocytes than did recipients of wild-type bone marrow. The Ly6C- monocytes remaining in the bone marrow of Nr4a1-/- mice were arrested in S phase of the cell cycle and underwent apoptosis. Thus, NR4A1 functions as a master regulator of the differentiation and survival of 'patrolling' Ly6C- monocytes.

CD4(+) regulatory T cells in a cynomolgus macaque model of Mycobacterium tuberculosis infection.

BACKGROUND: Mycobacterium tuberculosis infection in humans results in either latent infection or active tuberculosis. We sought to determine whether a higher frequency of regulatory T (T(reg)) cells predispose an individual toward active disease or whether T(reg) cells develop in response to active disease. METHODS: In cynomolgus macaques infected with a low dose of M. tuberculosis, approximately 50% develop primary tuberculosis, and approximately 50% become latently infected. Forty-one animals were monitored for 6-8 months to assess the correlation of the frequency of Foxp3(+) cells in peripheral blood and airways with the outcome of infection. RESULTS: In all animals, the frequency of T(reg) cells (CD4(+)Foxp3(+)) in peripheral blood rapidly decreased and simultaneously increased in the airways. Latently infected monkeys had a significantly higher frequency of T(reg) cells in peripheral blood before infection and during early infection, compared with monkeys that developed active disease. Monkeys with active disease experienced increased frequencies of T(reg) cells among peripheral blood mononuclear cells as they developed disease. CONCLUSIONS: Our data suggest that increased frequencies of T(reg) cells in active disease occur in response to increased inflammation rather than act as a causative factor in progression to active disease.

Aberrant TGF-beta signaling reduces T regulatory cells in ICAM-1-deficient mice, increasing the inflammatory response to Mycobacterium tuberculosis.

Foxp3+ T regulatory cells are required to prevent autoimmune disease, but also prevent clearance of some chronic infections. While natural T regulatory cells are produced in the thymus, TGF-beta1 signaling combined with T-cell receptor signaling induces the expression of Foxp3 in CD4+ T cells in the periphery. We found that ICAM-1-/- mice have fewer T regulatory cells in the periphery than WT controls, due to a role for ICAM-1 in induction of Foxp3 expression in response to TGF-beta1. Further investigation revealed a functional deficiency in the TGF-beta1-induced translocation of phosphorylated Smad3 from the cytoplasmic compartment to the nucleus in ICAM-1-deficient mice. This impairment in the TGF-beta1 signaling pathway is most likely responsible for the decrease in T regulatory cell induction in the absence of ICAM-1. We hypothesized that in the presence of an inflammatory response, reduced production of inducible T regulatory cells would be evident in ICAM-1-/- mice. Indeed, following Mycobacterium tuberculosis infection, ICAM-1-/- mice had a pronounced reduction in T regulatory cells in the lungs compared with control mice. Consequently, the effector T-cell response and inflammation were greater in the lungs of ICAM-1-/- mice, resulting in morbidity due to overwhelming pathology.

IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection.

IL-17 is a cytokine produced by T cells in response to IL-23. Recent data support a new subset of CD4 Th cells distinct from Th1 or Th2 cells that produce IL-17 and may contribute to inflammation. In this study, we demonstrate that, in naive mice, as well as during Mycobacterium tuberculosis infection, IL-17 production is primarily from gammadelta T cells and other non-CD4(+)CD8(+) cells, rather than CD4 T cells. The production of IL-17 by these cells is stimulated by IL-23 alone, and strongly induced by the cytokines, including IL-23, produced by M. tuberculosis-infected dendritic cells. IL-23 is present in the lungs early in infection and the IL-17-producing cells, such as gammadelta T cells, may represent a central innate protective response to pulmonary infection.

Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide.

The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-kappa B activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.