Bi-Sheng Liu

IL-29 and IFNα differ in their ability to modulate IL-12 production by TLR-activated human macrophages and exhibit differential regulation of the IFNγ receptor expression.

The interferon-λ (IFNλ) family of cytokines, consisting of interleukin-28A (IFNλ2), IL-28B (IFNλ3), and IL-29 (IFNλ1), have been extensively studied for their antiviral activities. However, little is known about the effect of IFNλ on antigen-presenting cells. In the present study, we show for the first time that IL-29 can increase Toll-like receptor (TLR)-induced IL-12p40 production by human monocyte-derived macrophages. In contrast, IL-29 did not affect monocytes or monocyte-derived dendritic cells (DCs) because of restricted IL-28 receptor α chain expression by macrophages. Furthermore, IL-29-treated macrophages were more responsive to IFNγ, because IL-29 enhanced IFNγ-induced IL-12p40 and tumor necrosis factor (TNF) production by macrophages on R848 stimulation. However, IFNα suppressed IFNγ-induced IL-12p40 and tumor necrosis factor TNF production by human macrophages. The differential effects of IL-29 and IFNα on the responsiveness of macrophages to IFNγ could not be explained by an effect on TLR7 or TLR8 mRNA expression or by altered IL-10 signaling. However, we demonstrated that IL-29 up-regulated, whereas IFNα down-regulated, the surface expression of the IFNγ receptor 1 chain on macrophages, thereby resulting in differential responsiveness of TLR-challenged macrophages to IFNγ. Our findings on the differences between IFNα and IL-29 in modulating TLR-induced cytokine production by macrophages may contribute to understanding the role of IFNs in regulating immunity to pathogens.

Modulation of dendritic cell function by persistent viruses

Worldwide, chronic viral infections cause major health problems with severe morbidity and mortality. HIV and hepatitis C virus (HCV) manifest themselves as persistent infections, but they are entirely distinct viruses with distinct replication mechanisms, tropism, and kinetics. Coinfections with HCV among people with HIV are emerging as a growing problem. Cellular immune responses play an important role in viral clearance and disease pathogenesis. However, cellular immunity to HIV and HCV is affected severely in chronic patients. Various hypotheses have been proposed to explain the dysfunctional T cell response, including viral escape mutations, exhaustion of the T cell compartment, and the activity of regulatory T cells. Also, modulation of the function of dendritic cells (DC) has been suggested as one of the mechanisms used by persistent viruses to evade the immune system. In this review, we will focus on DC interactions with one murine persistent virus (lymphocytic choriomeningitis virus clone 13) and two human persistent viruses (HIV‐1 and HCV), intending to examine if general strategies are used by persistent viruses to modulate the function of DC to improve our understanding of the mechanisms underlying the development and maintenance of viral persistence.

Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections

The consequences of chronic infection with the HCV on immunity to distinct pathogens are not fully appreciated, despite the potent modulatory effects of HCV on the immune system. We observed that upon TLR4 ligation, monocytes from chronic HCV patients demonstrated three to five times lower TNF and IL‐12p40 production as compared with healthy individuals. However, augmented production of TNF, IL‐12p40, and IL‐12p70 by monocytes was observed upon stimulation with R848. Importantly, we observed that the levels of IL‐10 in chronic HCV patients are higher in serum and that more IL‐10 is produced by monocytes as compared with healthy individuals. The inhibitory effect of IL‐10 on the production of proinflammatory cytokines by monocytes was only observed upon LPS stimulation but not upon R848 stimulation, showing that only the TLR4 pathway in monocytes is sensitive to the suppressive effects of IL‐10. Interestingly, monocytes stimulated with the TLR4 agonist, but not TLR8 agonist, produced higher levels of IL‐10 when exposed to patient serum as compared with serum from healthy individuals. Our results indicate that by differentially affecting TLR4 and TLR8 pathways, IL‐10 may mediate highly selective modulation of the function of monocytes observed in chronic HCV patients. This suggests that there is no overall increased susceptibility to pathogens but a specific suppression of the functionality of TLR4 signaling pathway in monocytes, which is, at least partly, mediated via IL‐10.

Type I and III interferons enhance IL-10R expression on human monocytes and macrophages, resulting in IL-10-mediated suppression of TLR-induced IL-12.

Currently, only about 30–50% of chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) patients respond to IFN‐based therapy. It has been suggested that IL‐10 is involved in suppressing the activity of type I IFNs on antigen‐presenting cells (APCs). However, the interaction between type I IFNs and IL‐10 is still not clear. Here we report that IFN‐α priming upregulated the expression of IL‐10R1 on monocytes, and subsequently IL‐10 induced a higher level of STAT3 phosphorylation in IFN‐primed cells. This indicates that IFN‐α increased the sensitivity of monocytes to IL‐10, and as a result, TLR‐induced IL‐12p70 by IFN‐pretreated cells was suppressed. Interestingly, both IFN‐β and IL‐29, a member of the type III IFN family, comparably sensitized monocytes and macrophages to IL‐10 stimulation, indicating a general effect of IFN on the activity of IL‐10 in APCs. In summary, we demonstrate that one of the consequences of priming human APCs with IFN is to promote the cells’ sensitivity to IL‐10, which leads to the inhibition of TLR‐induced IL‐12p70 production. Therefore, type I and III IFNs induce a suboptimal activation of immune cells. These findings are relevant for the development of strategies to further improve IFN‐based therapy for patients with multiple sclerosis or viral hepatitis.

IFN-λ-mediated IL-12 production in macrophages induces IFN-γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.

Potent immune activation in chronic hepatitis C patients upon administration of an oral inducer of endogenous interferons that acts via Toll-like receptor 7

BACKGROUNDnANA773, an oral prodrug of a small-molecule Toll-like receptor (TLR)7 agonist, induces a dose-related decrease in serum HCV RNA levels in chronic hepatitis C patients.nnnMETHODSnThe prodrug ANA773 was administered to healthy individuals and chronic hepatitis C patients. At different time points during the course of treatment, modulation of the phenotype and function of peripheral leukocytes were evaluated to determine the role of distinct immune cells on the clinical outcome of therapy.nnnRESULTSnEarly after administration of the TLR7 agonist, a mild transient reduction of the number of lymphocytes was observed in both healthy individuals and chronic hepatitis C patients. Moreover, repeated administration of ANA773 resulted in transiently reduced numbers of myeloid and plasmacytoid dendritic cells (DC) in blood. Interestingly, reduced plasmacytoid DC numbers as well as increased serum interferon (IFN)-α and IFN-γ inducible protein (IP)-10 levels were observed only in virological responders (≥1 log(10) IU/ml reduction of HCV RNA levels upon ANA773 treatment), but were absent in virological non-responders. In vitro stimulation of peripheral blood mononuclear cells from virological responders showed a high frequency of IFN-α-producing plasmacytoid DC upon stimulation in vitro with ANA773, whereas no IFN-α was induced in non-responders.nnnCONCLUSIONSnThese findings indicate that the viral load decline in chronic hepatitis C patients treated with the TLR7 agonist ANA773 is likely due to intrinsic differences in the induction of endogenous IFNs and IFN-stimulated gene products (IFN-α and IP-10) upon TLR7 ligation.

IFN-λ is able to augment TLR-mediated activation and subsequent function of primary human B cells

During the past decade, increased emphasis has been placed on finding alternatives to IFN‐α‐based therapies. One such alternative, IFN‐λ, has shown therapeutic promise in a variety of diseases, but research of this family of cytokines has been primarily focused on their antiviral activities. The goal of the present study was to investigate the role of IFN‐λ in the regulation and modulation of B cell function. We show that, similar to IFN‐α, IFN‐λ1 is able to augment TLR‐mediated B cell activation, partially attributed to an upregulation of TLR7 expression, and that both naϊve and memory B cells express the limiting type III IFN receptor component, IFN‐λR1. Furthermore, this IFN‐λ‐enhanced B cell activation resulted in increased cytokine and Ig production during TLR7 challenge, most prominently after the addition of helper T cell signals. Ultimately, these elevated cytokine and Ig levels could be partially attributed to the increase in proliferation of TLR7‐challenged B cells by both type I and type III IFNs. These findings demonstrate the ability of IFN‐λ to boost humoral immunity, an important attribute to consider for further studies on immunity to pathogens, vaccine development, and ongoing advancement of therapeutic strategies aimed at replacing IFN‐α‐based treatments with IFN‐λ.

The response to TLR ligation of human CD16(+)CD14(-) monocytes is weakly modulated as a consequence of persistent infection with the hepatitis C virus

Little is known about the frequency and function of CD16(+)CD14(-) monocytes from chronic HCV patients. We observed that the absolute numbers and ratio of CD16(+)CD14(-) to CD14(+)CD16(-) monocytes were similar between chronic HCV patients and healthy individuals. Functionally, we found that CD16(+)CD14(-) monocytes are more responsive to TLR8-ligation and only weakly responsive to LPS stimulation in producing TNF as compared to CD14(+)CD16(-) monocytes. We found no overt impairment of the function of CD16(+)CD14(-) monocytes from patients, except for an augmented induction of MIP-1β-producing CD16(+)CD14(-) monocytes upon TLR4-ligation. However, the increased frequency of MIP-1β-producing CD16(+)CD14(-) monocytes was not associated with viral load, ALT or fibrosis level. Our findings indicate that, different from other infectious diseases, the frequency and function of CD16(+)CD14(-) monocytes are only minimally altered as a consequence of the persistent state of HCV infections, and our findings therefore do not suggest a role for CD16(+)CD14(-) monocytes in HCV pathogenesis.

Analysis of the transcriptome and immune function of monocytes during IFNα-based therapy in chronic HCV revealed induction of TLR7 responsiveness

Although in vitro studies have been performed to dissect the mechanism of action of IFNα, detailed in vivo studies on the long-term effects of IFNα on monocytes have not been performed. Here we examined peripheral blood from 14 chronic HCV patients at baseline and 12 weeks after start of IFNα-based therapy. Monocytes were phenotyped by flow-cytometry and their function evaluated upon TLR stimulation and assessed by multiplex cytokine assays. During therapy of HCV patients, monocytes displayed a hyperactive state as evidenced by increased TLR-induced pro-inflammatory cytokine levels, as well as enhanced CD69 and CD83 mRNA and protein expression. Moreover, monocytes from 8 patients at baseline and 12 weeks after start of IFNα-based therapy were transcriptomically profiled by high throughput RNA-sequencing. Detailed RNA-seq analysis of monocytes showed significant ISG mRNA induction during therapy. Importantly, IFNα-based therapy activated TLR7 signaling pathways, as demonstrated by up-regulated expression of TLR7, MyD88, and IRF7 mRNA, whereas other TLR family members as well as CD1c, CLEC4C, and CLEC9A were not induced. The induction of TLR7 responsiveness of monocytes by IFNα in vivo in HCV patients is relevant for the development of TLR7 agonists that are currently under development as a promising immunotherapeutic compounds to treat chronic viral hepatitis.