Jonathan P. Moorman

Tim-3 Pathway Controls Regulatory and Effector T Cell Balance during Hepatitis C Virus Infection

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3+ Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3+ Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2–producing CD4+CD25+Foxp3− Teffs, but also on CD4+CD25+Foxp3+ Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3+ Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2–producing Teffs. Moreover, Foxp3+ Tregs were found to be more resistant to, and Foxp3− Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4+CD25+ T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3+ Tregs/Foxp3− Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.

Tim-3 Negatively Regulates IL-12 Expression by Monocytes in HCV Infection

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a newly identified negative immunomodulator that is up-regulated on dysfunctional T cells during viral infections. The expression and function of Tim-3 on human innate immune responses during HCV infection, however, remains poorly characterized. In this study, we report that Tim-3 is constitutively expressed on human resting CD14+ monocyte/macrophages (M/MØ) and functions as a cap to block IL-12, a key pro-inflammatory cytokine linking innate and adaptive immune responses. Tim-3 expression is significantly reduced and IL-12 expression increased upon stimulation with Toll-like receptor 4 (TLR4) ligand - lipopolysaccharide (LPS) and TLR7/8 ligand - R848. Notably, Tim-3 is over-expressed on un-stimulated as well as TLR-stimulated M/MØ, which is inversely associated with the diminished IL-12 expression in chronically HCV-infected individuals when compared to healthy subjects. Up-regulation of Tim-3 and inhibition of IL-12 are also observed in M/MØ incubated with HCV-expressing hepatocytes, as well as in primary M/MØ or monocytic THP-1 cells incubated with HCV core protein, an effect that mimics the function of complement C1q and is reversible by blocking the HCV core/gC1qR interaction. Importantly, blockade of Tim-3 signaling significantly rescues HCV-mediated inhibition of IL-12, which is primarily expressed by Tim-3 negative M/MØ. Tim-3 blockade reduces HCV core-mediated expression of the negative immunoregulators PD-1 and SOCS-1 and increases STAT-1 phosphorylation. Conversely, blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through crosstalk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.

Cross-Talk between Programmed Death-1 and Suppressor of Cytokine Signaling-1 in Inhibition of IL-12 Production by Monocytes/Macrophages in Hepatitis C Virus Infection

Hepatitis C virus (HCV) dysregulates innate immune responses and induces persistent viral infection. We previously demonstrated that HCV core protein impairs IL-12 expression by monocytes/macrophages (M/MΦs) through interaction with a complement receptor gC1qR. Because HCV core-mediated lymphocyte dysregulation occurs through the negative immunomodulators programmed death-1 (PD-1) and suppressor of cytokine signaling-1 (SOCS-1), the aim of this study was to examine their role in HCV core-mediated IL-12 suppression in M/MΦs. We analyzed TLR-stimulated, primary CD14+ M/MΦs from chronically HCV-infected and healthy subjects or the THP-1 cell line for PD-1, SOCS-1, and IL-12 expression following HCV core treatment. M/MΦs from HCV-infected subjects at baseline exhibited comparatively increased PD-1 expression that significantly correlated with the degree of IL-12 inhibition. M/MΦs isolated from healthy and HCV-infected individuals and treated with HCV core protein displayed increased PD-1 and SOCS-1 expression and decreased IL-12 expression, an effect that was also observed in cells treated with gC1qR’s ligand, C1q. Blocking gC1qR rescued HCV core-induced PD-1 upregulation and IL-12 suppression, whereas blocking PD-1 signaling enhanced IL-12 production and decreased the expression of SOCS-1 induced by HCV core. Conversely, silencing SOCS-1 expression using small interfering RNAs increased IL-12 expression and inhibited PD-1 upregulation. PD-1 and SOCS-1 were found to associate by coimmunoprecipitation studies, and blocking PD-1 or silencing SOCS-1 in M/MΦ led to activation of STAT-1 during TLR-stimulated IL-12 production. These data suggested that HCV core/gC1qR engagement on M/MΦs triggers the expression of PD-1 and SOCS-1, which can associate to deliver negative signaling to TLR-mediated pathways controlling expression of IL-12, a key cytokine linking innate and adaptive immunity.

Differential regulation of SOCS-1 signalling in B and T lymphocytes by hepatitis C virus core protein.

Hepatitis C virus (HCV) infection is characterized by a strong propensity toward chronicity, autoimmune phenomena and lymphomagenesis, supporting a role for lymphocyte dysregulation during persistent viral infection. We have shown that HCV core protein inhibits T‐cell functions through interaction with a complement receptor, gC1qR. Here, we further report that B cells also express gC1qR that can be bound by HCV core protein. Importantly, using flow cytometry, we demonstrated differential regulation of B and T lymphocytes by the HCV core–gC1qR interaction, with down‐regulation of CD69 activation in T cells but up‐regulation of CD69 activation and cell proliferation in B cells. HCV core treatment led to decreased interferon‐γ production in CD8+ T cells but to increased immunoglobulin M and immunoglobulin G production as well as cell surface expression of costimulatory and chemokine receptors, including CD86 (B7‐2), CD154 (CD40L) and CD195 (CCR5), in CD20+ B cells. Finally, we showed down‐regulation of suppressor of cytokine signalling‐1 (SOCS‐1) using real‐time reverse transcription–polymerase chain reaction, accompanied by up‐regulation of signal transducer and activator of transcription‐1 (STAT1) phosphorylation in B cells in response to HCV core protein, with the opposite pattern observed in HCV core‐treated T cells. This study demonstrates differential regulation of B and T lymphocytes by HCV core and supports a mechanism by which lymphocyte dysregulation occurs in the course of persistent HCV infection.

The C-terminal region of hepatitis C core protein is required for Fas-ligand independent apoptosis in Jurkat cells by facilitating Fas oligomerization.

Hepatitis C virus (HCV) is remarkable for its ability to establish persistent infection. Studies suggest that HCV core protein modulates immune responses to viral infection and can bind Fas receptor in vitro. To further examine the role of HCV core protein in Fas signaling, full-length (aa 1-192) and truncated (aa 1-152) HCV core proteins were expressed in Jurkat lymphocytes and cells were assayed for apoptotic response, caspase activation, and Fas activation. Jurkat expressing full-length but not truncated core protein exhibited ligand-independent apoptosis. Cytoplasmic targeting of truncated core protein recapitulated its ability to induce apoptosis. Activation of caspases 8 and 3 was necessary and sufficient for full-length core to induce apoptosis. Jurkat cells expressing full-length but not truncated core protein induced Fas receptor aggregation. HCV core activates apoptotic pathways in Jurkat via Fas and requires cytoplasmic localization of core. Infection of host lymphocytes by HCV may alter apoptotic signaling and skew host responses to acute infection.

PD-1 negatively regulates interleukin-12 expression by limiting STAT-1 phosphorylation in monocytes/macrophages duringchronic hepatitis C virus infection

Hepatitis C virus (HCV) is remarkably efficient at evading host immunity to establish chronic infection. During chronic HCV infection, interleukin‐12 (IL‐12) produced by monocytes/macrophages (M/Mφ) is significantly suppressed. Programmed death‐1 (PD‐1), an inhibitory receptor on immune cells, plays a pivotal role in suppressing T‐cell responses during chronic viral infection. To determine whether PD‐1 regulates IL‐12 production by M/Mφ during chronic HCV infection, we examined the expressions of PD‐1, its ligand PDL‐1, and their relationship with IL‐12 production in M/Mφ from HCV‐infected, HCV‐resolved, and healthy subjects by flow cytometry. Toll‐like receptor (TLR) ‐mediated IL‐12 production by M/Mφ was selectively suppressed, while PD‐1/PDL‐1 expressions were up‐regulated, in HCV‐infected subjects compared with HCV‐resolved or healthy subjects. Up‐regulation of PD‐1 was inversely associated with the degree of IL‐12 inhibition in HCV infection. Interestingly, the reduced response of M/Mφ from HCV‐infected individuals to TLR ligands appeared not to be the result of a lack of the ability to sense pathogen, but to an impaired activation of intracellular janus kinase/signal transducer and activator of transfection (STAT) pathway as represented by inhibited STAT‐1 phosphorylation in M/Mφ from HCV‐infected individuals compared with HCV‐negative subjects. Successful HCV treatment with pegylated interferon/ribavirin or blocking PD‐1/PDL‐1 engagement ex vivo led to reduced PD‐1 expression and improved IL‐12 production as well as STAT‐1 activation in M/Mφ from HCV‐infected individuals. These results suggest that the PD‐1 inhibitory pathway may negatively regulate IL‐12 expression by limiting STAT‐1 phosphorylation in M/Mφ during chronic HCV infection.

Impaired hepatitis B vaccine responses during chronic hepatitis C infection: involvement of the PD-1 pathway in regulating CD4+ T cell responses

Vaccination for hepatitis B virus (HBV) in the setting of hepatitis C virus (HCV) infection is recommended, but responses to vaccination are blunted when compared to uninfected populations. The mechanism for this failure of immune response in HCV-infected subjects remains unknown but is thought to be a result of lymphocyte dysfunction during chronic viral infection. We have recently demonstrated that PD-1, a novel negative immunomodulator for T cell receptor (TCR) signaling, is involved in T and B lymphocyte dysregulation during chronic HCV infection. In this report, we further investigated the role of the PD-1 pathway in regulation of CD4(+) T cell responses to HBV vaccination in HCV-infected individuals. In a prospective HCV infected cohort, a poor response rate to HBV vaccination as assayed by seroconversion was observed in HCV-infected subjects (53%), while a high response rate was observed in healthy or spontaneously HCV-resolved individuals (94%). CD4(+) T cell responses to ex vivo stimulations of anti-CD3/CD28 antibodies or hepatitis B surface antigen (HBsAg) were found to be lower in HBV vaccine non-responders compared to those responders in HCV-infected individuals who had received a series of HBV immunizations. PD-1 expression on CD4(+) T cells was detected at relatively higher levels in these HBV vaccine non-responders than those who responded, and this was inversely associated with the cell activation status. Importantly, blocking the PD-1 pathway improved T cell activation and proliferation in response to ex vivo HBsAg or anti-CD3/CD28 stimulation in HBV vaccine non-responders. These results suggest that PD-1 signaling may be involved in impairing CD4(+) T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this negative signaling pathway might improve success rates of immunization in the setting of chronic viral infection.

PD-1 modulates regulatory T cells and suppresses T-cell responses in HCV-associated lymphoma

T regulatory (TR) cells suppress T‐cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death‐1 (PD‐1) also has a pivotal role in regulation of T‐cell functions during chronic viral infection. To examine the role of PD‐1 pathway in regulating TR‐cell functions that inhibit T‐cell responses during virus‐associated malignancy, TR cells were investigated in the setting of hepatitis C virus‐associated lymphoma (HCV‐L), non‐HCV‐associated lymphoma (non‐HCV‐L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4+CD25+ and CD8+CD25+ TR cells, as well as high levels of PD‐1 expressions on these TR cells were found in the peripheral blood of subjects with HCV‐L compared with those from non‐HCV‐L or HCV alone or HS. TR cells from the HCV‐L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of TR cells in peripheral blood mononuclear cells from HCV‐L improved T‐cell proliferation. Additionally, the suppressed T‐cell activation and proliferation in HCV‐L was partially restored by blocking the PD‐1 pathway ex vivo, resulting in both a reduction in TR‐cell number and the ability of TR to suppress the activity of effector T cells. This study suggests that the PD‐1 pathway is involved in regulating TR cells that suppress T‐cell functions in the setting of HCV‐associated B‐cell lymphoma.

Myeloid-derived Suppressor Cells: Paradoxical Roles in Infection and Immunity

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature suppressor cells that are generated due to aberrant myelopoiesis under pathological conditions. Although MDSCs have been recognized for more than 20 years under the guise of different monikers, these particular populations of myeloid cells gained more attention recently due to their immunosuppressive properties, which halt host immune responses to growing cancers or overwhelming infections. While MDSCs may contribute to immune homeostasis after infection or tissue injury by limiting excessive inflammatory processes, their expansion may be at the expense of pathogen elimination and thus may lead to disease persistence. Therefore, MDSCs may be either damaging or obliging to the host by attenuating, for example, antitumor or anti-infectious immune responses. In this review, we recapitulate the biological and immunological aspects of MDSCs, including their generation, distribution, trafficking and the factors involved in their activation, expansion, suppressive functions, and interplay between MDSCs and regulatory T cells, with a focus on the perspectives of infection and inflammation.

C-reactive protein-bound enzymatically modified low-density lipoprotein does not transform macrophages into foam cells

The formation of low-density lipoprotein (LDL) cholesterol-loaded macrophage foam cells contributes to the development of atherosclerosis. C-reactive protein (CRP) binds to atherogenic forms of LDL, but the role of CRP in foam cell formation is unclear. In this study, we first explored the binding site on CRP for enzymatically modified LDL (E-LDL), a model of atherogenic LDL to which CRP binds. As reported previously, phosphocholine (PCh) inhibited CRP-E-LDL interaction, indicating the involvement of the PCh-binding site of CRP in binding to E-LDL. However, the amino acids Phe66 and Glu81 in CRP that participate in CRP-PCh interaction were not required for CRP-E-LDL interaction. Surprisingly, blocking of the PCh-binding site with phosphoethanolamine (PEt) dramatically increased the binding of CRP to E-LDL. The PEt-mediated enhancement in the binding of CRP to E-LDL was selective for E-LDL because PEt inhibited the binding of CRP to another PCh-binding site-ligand pneumococcal C-polysaccharide. Next, we investigated foam cell formation by CRP-bound E-LDL. We found that, unlike free E-LDL, CRP-bound E-LDL was inactive because it did not transform macrophages into foam cells. The function of CRP in eliminating the activity of E-LDL to form foam cells was not impaired by the presence of PEt. Combined data lead us to two conclusions. First, PEt is a useful compound because it potentiates the binding of CRP to E-LDL and, therefore, increases the efficiency of CRP to prevent transformation of macrophages into E-LDL-loaded foam cells. Second, the function of CRP to prevent formation of foam cells may influence the process of atherogenesis.