The Human Male Liver Is Predisposed to Inflammation Via Enhanced Myeloid Responses to Inflammatory Triggers.

Background & Aim: Men have a higher prevalence of liver disease. Liver myeloid cells can regulate tissue inflammation, which drives progression of liver disease. We hypothesized that sex alters the responsiveness of liver myeloid cells, predisposing men to severe liver inflammation. Methods: Luminex was done on plasma from Hepatitis B Virus infected patients undergoing nucleoside analogue cessation in 45 male and female patients. We collected immune cells from the sinusoids of uninfected livers of 53 male and female donors. Multiparametric flow cytometry was used to phenotype and characterize immune composition. Isolated monocytes were stimulated with TLR ligands to measure the inflammatory potential and the expression of regulators of TLR signaling. Results: We confirmed that men experienced more frequent and severe liver damage upon Hepatitis B Virus reactivation, which was associated with inflammatory markers of myeloid activation. No differences were observed in the frequency or phenotype of sinusoidal myeloid cells between male and female livers. However, monocytes from male livers produced more inflammatory cytokines and chemokines in response to TLR stimulation than female monocytes. We investigated negative regulators of TLR signaling and found that TOLLIP was elevated in female liver-derived monocytes. Conclusions: Our data show that enhanced responsiveness of myeloid cells from the male liver predisposes men to inflammation, which was associated with altered expression of negative regulators of TLR signaling.

A human recombinant analogue to plasma-derived vaccinia immunoglobulin prophylactically and therapeutically protects against lethal orthopoxvirus challenge.

Orthopoxviruses such as variola and monkeypox viruses continue to threaten the human population. Monkeypox virus is endemic in central and western Africa and outbreaks have reached as far as the U.S. Although variola virus, the etiologic agent of smallpox, has been eradicated by a successful vaccination program, official and likely clandestine stocks of the virus exist. Moreover, studies with ectromelia virus (the etiological agent of mousepox) have revealed that IL-4 recombinant viruses are significantly more virulent than wild-type viruses even in mice treated with vaccines and/or antivirals. For these reasons, it is critical that antiviral modalities are developed to treat these viruses should outbreaks, or deliberate dissemination, occur. Currently, 2 antivirals (brincidofovir and tecovirimat) are in the U.S. stockpile allowing for emergency use of the drugs to treat smallpox. Both antivirals have advantages and disadvantages in a clinical and emergency setting. Here we report on the efficacy of a recombinant immunoglobulin (rVIG) that demonstrated efficacy against several orthopoxviruses in vitro and in vivo in both a prophylactic and therapeutic fashion. A single intraperitoneal injection of rVIG significantly protected mice when given up to 14 days before or as late as 6 days post challenge. Moreover, rVIG reduced morbidity, as measured by weight-change, as well as several previously established biomarkers of disease. In rVIG treated mice, we found that vDNA levels in blood were significantly reduced, as was ALT (a marker of liver damage) and infectious virus levels in the liver. No apparent adverse events were observed in rVIG treated mice, suggesting the immunoglobulin is well tolerated. These findings suggest that recombinant immunoglobulins could be candidates for further evaluation and possible licensure under the FDA Animal Rule.

Selective estrogen receptor modulator, tamoxifen, inhibits Zika virus infection.

Zika virus (ZIKV) is an arbovirus belonging to the flaviviridae family with a risk assessment that has been increasing in recent years and was labeled a global health emergency by the World Health Organization in 2016. There are currently no Food and Drug Administration-approved treatment options available for ZIKV, so expeditious development of treatment options is urgent. To expedite this process, an on-market drug, tamoxifen (TAM), was selected as a promising candidate for repurposing due to its wide range of biological activities and because it has already been shown to possess activity against hepatitis C virus, a flavivirus in a separate genus. Anti-ZIKV activity of TAM was assessed by compound screens using an infectious virus and mechanistic details were gleaned from time of addition and virucidal studies. TAM and an active metabolite, 4-hydroxytamoxifen (TAM-OH), both showed promising antiviral activity (EC50 ≈9 and 5 µM, respectively) in initial compound screening and up to 8-h postinfection, though the virucidal assay indicated that they do not possess any direct virucidal activity. Additionally, TAM was assessed for its activity against ZIKV in the human male germ cell line, SEM-1, due to the sexually transmitted nature of ZIKV owing to its extended survival times in germ cells. Virus titers show diminished replication of ZIKV over 7 days compared to controls. These data indicate that TAM has the potential to be repurposed as an anti-ZIKV therapeutic and warrants further investigation.

IFN-α Suppresses Myeloid Cytokine Production, Impairing IL-12 Production and the Ability to Support T-Cell Proliferation.

BACKGROUND: Interferon-α (IFN-α) can suppress production of T-cell polarizing cytokines or induce inhibitory antigen-presenting cells that suppress T-cell activation. Previous studies showed that IFN-α therapy fails to boost virus-specific T-cell immunity in patients with chronic hepatitis B virus infection. Our aim was to determine whether IFN-α exposure alters human antigen-presenting cell function in vivo. METHODS: We investigated the immunomodulatory effects using peripheral blood mononuclear cells from healthy donors exposed to IFN-α and chronic hepatitis B (CHB) patients starting IFN-α therapy. RESULTS: IFN-α increased HLA-DR, CD80, CD86, and PD-L1 expression on healthy donor monocytes. In contrast to the activated phenotype, IFN-α inhibited Toll-like receptor-induced cytokine production and monocyte-induced T-cell proliferation. In CHB patients, peg-IFN treatment induced an interferon-stimulated gene signature in monocytes and increased HLA-DR, CD80, CD86, and PD-L1 expression. As early as 3 days after CHB patients started treatment, IFN-α inhibited monocyte cytokine production and T-cell stimulation ex vivo. IFN-α-mediated inhibition of IL-12 production, rather than inhibitory receptor expression, was responsible for inhibition of T-cell proliferation. Addition of IL-12 restored T-cell proliferation to baseline levels. CONCLUSIONS: Understanding how professional antigen-presenting cells respond to immunomodulation is important for both new innate and adaptive-targeted immunotherapies. CLINICAL TRIALS REGISTRATION: NCT00962871.

Type I Interferon Responses Drive Intrahepatic T cells to Promote Metabolic Syndrome.

Obesity-related insulin resistance is driven by low-grade chronic inflammation of metabolic tissues. In the liver, non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance and systemic glucose dysregulation. However, the immunological factors supporting these processes are poorly understood. We found that the liver accumulates pathogenic CD8+ T cell subsets which control hepatic insulin sensitivity and gluconeogenesis during diet-induced obesity in mice. In a cohort of human patients, CD8+ T cells represent a dominant intrahepatic immune cell population which links to glucose dysregulation. Accumulation and activation of these cells are largely supported by type I interferon (IFN-I) responses in the liver. Livers from obese mice upregulate critical interferon regulatory factors (IRFs), interferon stimulatory genes (ISGs), and IFNα protein, while IFNαR1-/- mice, or CD8-specific IFNαR1-/- chimeric mice are protected from disease. IFNαR1 inhibitors improve metabolic parameters in mice, while CD8+ T cells and IFN-I responses correlate with NAFLD activity in human patients. Thus, IFN-I responses represent a central immunological axis that governs intrahepatic T cell pathogenicity during metabolic disease.

Alveolar macrophage development in mice requires L-plastin for cellular localization in alveoli.

Alveolar macrophages are lung-resident sentinel cells that develop perinatally and protect against pulmonary infection. Molecular mechanisms controlling alveolar macrophage generation have not been fully defined. Here, we show that the actin-bundling protein L-plastin (LPL) is required for the perinatal development of alveolar macrophages. Mice expressing a conditional allele of LPL (CD11c.Crepos-LPLfl/fl) exhibited significant reductions in alveolar macrophages and failed to effectively clear pulmonary pneumococcal infection, showing that immunodeficiency results from reduced alveolar macrophage numbers. We next identified the phase of alveolar macrophage development requiring LPL. In mice, fetal monocytes arrive in the lungs during a late fetal stage, maturing to alveolar macrophages through a prealveolar macrophage intermediate. LPL was required for the transition from prealveolar macrophages to mature alveolar macrophages. The transition from prealveolar macrophage to alveolar macrophage requires the upregulation of the transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), which is induced by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite abundant lung GM-CSF and intact GM-CSF receptor signaling, PPAR-γ was not sufficiently upregulated in developing alveolar macrophages in LPL-/- pups, suggesting that precursor cells were not correctly localized to the alveoli, where GM-CSF is produced. We found that LPL supports 2 actin-based processes essential for correct localization of alveolar macrophage precursors: (1) transmigration into the alveoli, and (2) engraftment in the alveoli. We thus identify a molecular pathway governing neonatal alveolar macrophage development and show that genetic disruption of alveolar macrophage development results in immunodeficiency.

Dissecting the dendritic cell controversy in chronic hepatitis B virus infection.

Therapeutic vaccines to boost endogenous T-cell immunity rely on the stimulatory capacity of dendritic cells (DCs). The functionality of DCs in chronic hepatitis B virus (HBV) infection has been a long-standing debate. Therefore, we have attempted to summarize multiple studies investigating DC function in chronic HBV patients to determine whether common observations can be drawn. We found that the frequency and function of ex vivo-tested myeloid and plasmacytoid DCs were largely intact in patients with HBV infection and similar to those of healthy donor DCs. The main exception was reduced IFN-α production by plasmacytoid DC from chronic HBV patients. This reduced IFN-α production correlated with liver inflammation in multiple studies but not with viral load, suggesting that viral antigens have little effect on DC function. The majority of the confusion about DC function arises from studies reporting the reduced function of healthy donor DCs exposed to various sources of HBV in vitro. These direct effects of viral antigens are in contrast to data from HBV-infected patients. The variations in the assays used and areas that require further investigation are also covered.

The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

The cystine/glutamate antiporter regulates dendritic cell differentiation and antigen presentation.

The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.

Crosstalk between PKA and Epac regulates the phenotypic maturation and function of human dendritic cells.

The cAMP-dependent signaling pathways that orchestrate dendritic cell (DC) maturation remain to be defined in detail. Although cAMP was previously thought to signal exclusively through protein kinase A (PKA), it is now clear that cAMP also activates exchange protein activated by cAMP (Epac), a second major cAMP effector. Whether cAMP signaling via PKA is sufficient to drive DC maturation or whether Epac plays a role has not been examined. In this study, we used cAMP analogs to selectively activate PKA or Epac in human monocyte-derived DCs and examined the effect of these signaling pathways on several hallmarks of DC maturation. We show that PKA activation induces DC maturation as evidenced by the increased cell-surface expression of MHC class II, costimulatory molecules, and the maturation marker CD83. PKA activation also reduces DC endocytosis and stimulates chemotaxis to the lymph node-associated chemokines CXCL12 and CCL21. Although PKA signaling largely suppresses cytokine production, the net effect of PKA activation translates to enhanced DC activation of allogeneic T cells. In contrast to the stimulatory effects of PKA, Epac signaling has no effect on DC maturation or function. Rather, Epac suppresses the effects of PKA when both pathways are activated simultaneously. These data reveal a previously unrecognized crosstalk between the PKA and Epac signaling pathways in DCs and raise the possibility that therapeutics targeting PKA may generate immunogenic DCs, whereas those that activate Epac may produce tolerogenic DCs capable of attenuating allergic or autoimmune disease.

Ca2+-dependent calmodulin binding to FcRn affects immunoglobulin G transport in the transcytotic pathway.

The Fcgamma receptor FcRn transports immunoglobulin G (IgG) so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here, we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic alpha-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route.