Wendy R. Winnall

Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies

A better understanding of immunity to influenza virus is needed to generate cross-protective vaccines. Engagement of Ab-dependent cellular cytotoxicity (ADCC) Abs by NK cells leads to killing of virus-infected cells and secretion of antiviral cytokines and chemokines. ADCC Abs may target more conserved influenza virus Ags compared with neutralizing Abs. There has been minimal interest in influenza-specific ADCC in recent decades. In this study, we developed novel assays to assess the specificity and function of influenza-specific ADCC Abs. We found that healthy influenza-seropositive young adults without detectable neutralizing Abs to the hemagglutinin of the 1968 H3N2 influenza strain (A/Aichi/2/1968) almost always had ADCC Abs that triggered NK cell activation and in vitro elimination of influenza-infected human blood and respiratory epithelial cells. Furthermore, we detected ADCC in the absence of neutralization to both the recent H1N1 pandemic strain (A/California/04/2009) as well as the avian H5N1 influenza hemagglutinin (A/Anhui/01/2005). We conclude that there is a remarkable degree of cross-reactivity of influenza-specific ADCC Abs in seropositive humans. Targeting cross-reactive influenza-specific ADCC epitopes by vaccination could lead to improved influenza vaccines.

The regulation and functions of activin and follistatin in inflammation and immunity

The activins are members of the transforming growth factor β superfamily with broad and complex effects on cell growth and differentiation. Activin A has long been known to be a critical regulator of inflammation and immunity, and similar roles are now emerging for activin B, with which it shares 65% sequence homology. These molecules and their binding protein, follistatin, are widely expressed, and their production is increased in many acute and chronic inflammatory conditions. Synthesis and release of the activins are stimulated by inflammatory cytokines, Toll-like receptor ligands, and oxidative stress. The activins interact with heterodimeric serine/threonine kinase receptor complexes to activate SMAD transcription factors and the MAP kinase signaling pathways, which mediate inflammation, stress, and immunity. Follistatin binds to the activins with high affinity, thereby obstructing the activin receptor binding site, and targets them to cell surface proteoglycans and lysosomal degradation. Studies on transgenic mice and those with gene knockouts, together with blocking studies using exogenous follistatin, have established that activin A plays critical roles in the onset of cachexia, acute and chronic inflammatory responses such as septicemia, colitis and asthma, and fibrosis. However, activin A also directs the development of monocyte/macrophages, myeloid dendritic cells, and T cell subsets to promote type 2 and regulatory immune responses. The ability of both endogenous and exogenous follistatin to block the proinflammatory and profibrotic actions of activin A has led to interest in this binding protein as a potential therapeutic for limiting the severity of disease and to improve subsequent damage associated with inflammation and fibrosis. However, the ability of activin A to sculpt the subsequent immune response as well means that the full range of effects that might arise from blocking activin bioactivity will need to be considered in any therapeutic applications.

Rat resident testicular macrophages have an alternatively activated phenotype and constitutively produce interleukin-10 in vitro

The ability of the rodent testis to tolerate graft alloantigens and spermatogenic cell autoantigens is well known. The mechanisms underlying this “immune privilege” are poorly understood, but the numerous resident TMs have been implicated. Although it has been assumed that TMs display a phenotype consistent with immune privilege, this has not been formally established. Consequently, TMs were isolated from adult rats and cultured under basal conditions and following stimulation with LPS and IFN‐γ (classical activation) or IL‐4 (alternative activation). BMMs matured in vitro were used as control. Expression of the classical (proinflammatory) activation markers TNF‐α, IL‐1β, iNOS, IL‐6, RANTES, IL‐12p40, and SOCS3 and alternative (immunoregulatory) activation markers IL‐10, TGF‐β1, CXCL2, and SOCS1 was measured by QPCR or ELISA. In culture, TMs were characterized by poor expression of classical activation genes and TGF‐β1 but constitutively high IL‐10 production and reduced costimulatory activity in a polyclonal T cell activation assay. This pattern of gene expression was associated with TMs expressing the scavenger receptor CD163, which is characteristic of tissue resident macrophages and alternative activation. By contrast, CD163‐negative TMs displayed reduced inflammatory gene expression but did not constitutively produce IL‐10. These data indicate that under the influence of the testicular environment, macrophages adopt an alternatively activated phenotype, involving reduced capacity for proinflammatory gene expression, constitutive IL‐10 production, and impaired ability to support T cell activation, consistent with a role in maintaining testicular immune privilege.

Differential responses of epithelial Sertoli cells of the rat testis to Toll-Like receptor 2 and 4 ligands: implications for studies of testicular inflammation using bacterial lipopolysaccharides

The relative contribution of epithelial Sertoli cells in response to bacterial infection of the testis remains poorly characterised, since studies on inflammatory properties of these cells have invariably used unpurified lipopolysaccharide (LPS) preparations contaminated with bacterial lipopeptides. Consequently, isolated rat Sertoli cells were stimulated with either unextracted or phenol re-extracted LPS, and analysed for Toll-like receptor (TLR) 4, TLR2 and inflammatory cytokine gene expression by quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of TLR4 and its co-receptor protein myeloid differentiation (MD) 2 in Sertoli cells and testicular macrophages were similar, but Sertoli cells displayed low basal or LPS-induced expression of the TLR4 accessory protein, CD14. In Sertoli cells, unextracted LPS produced cytokine responses which were considerably greater in magnitude and duration compared with their response to purified LPS. Sertoli cells also responded to the synthetic lipopeptide, Pam3Cys (a TLR2 ligand) with a similar pattern of prolonged gene expression. Sertoli cells were more than 10-fold less sensitive to purified LPS than macrophages, but expressed similar levels of interleukin (IL)-1α and IL-6, and much greater levels of the immunoregulatory cytokine activin A, when maximally stimulated. These data demonstrate that Sertoli cells display differential cytokine responses to bacterial stimuli, mediated by both TLR2 and TLR4, that are distinct from those of testicular macrophages.

Regulation of activin and inhibin in the adult testis and the evidence for functional roles in spermatogenesis and immunoregulation.

Activin A provides a unique link between reproduction and immunity, which is especially significant in the adult testis. This cytokine, together with inhibin B and follistatin acting as regulators of activin A activity, is fundamentally involved in the regulation of spermatogenesis and testicular steroidogenesis. However, activin A also has a much broader role in control of inflammation, fibrosis and immunity. In the Sertoli cell, activin A is regulated by signalling pathways that normally regulate stress and inflammation, signalling pathways that intersect with the classical hormonal regulatory pathways mediated by FSH. Modulation of activin A production and activity during spermatogenesis is implicated in the fine control of the cycle of the seminiferous epithelium. The immunoregulatory properties of activin A also suggest that it may be involved in maintaining testicular immune privilege. Consequently, elevated activin A production within the testis during inflammation and infection may contribute to spermatogenic failure, fibrosis and testicular damage.

Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity in Intravenous Immunoglobulin as a Potential Therapeutic Against Emerging Influenza Viruses

BACKGROUND Intravenous immunoglobulin (IVIG) is a purified pool of human antibodies from thousands of donors that is used to prevent or treat primary immune deficiency, several infectious diseases, and autoimmune diseases. The antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against heterologous influenza strains may be present in IVIG preparations. METHODS We tested 8 IVIG preparations prior to the 2009 H1N1 swine-origin influenza pandemic and 10 IVIG preparations made after 2010 for their ability to mediate influenza-specific ADCC. RESULTS ADCC mediating antibodies to A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) were detected in IVIG preparations prior to the 2009-H1N1 pandemic. The HA-specific ADCC targeted both the HA1 and HA2 regions of A(H1N1)pdm09 HA and was capable of recognizing a broad range of HA proteins including those from recent avian influenza strains A(H5N1) and A(H7N9). The low but detectable ADCC recognition of A(H7N9) was likely due to rare individuals in the population contributing cross-reactive antibodies to IVIG. CONCLUSIONS IVIG preparations contain broadly cross-reactive ADCC mediating antibodies. IVIG may provide at least some level of protection for individuals at high risk of severe influenza disease, especially during influenza pandemics prior to the development of effective vaccines.

Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.

BACKGROUND During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed. METHODS We tested serum specimens obtained from 182 individuals aged 1-72 years that were collected either immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein. RESULTS A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals aged >45 years (28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged 1-14 years (11/31) and 15-45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza virus-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared with younger individuals. CONCLUSIONS ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009 A(H1N1) pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.

Constitutive Expression of Prostaglandin-Endoperoxide Synthase 2 by Somatic and Spermatogenic Cells Is Responsible for Prostaglandin E2 Production in the Adult Rat Testis

Abstract Prostaglandins (PGs), particularly PGE2, have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE2 measurements by ELISA. The mRNA for both the “constitutive” Ptgs1 and the “inducible” Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE2 in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE2 production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE2 but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE2; and 3) testicular PTGS2 expression and intratesticular PGE2 levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the “inducible” PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.

Tumour necrosis factor-α stimulates human neutrophils to release preformed activin A

Activin A, a member of the transforming growth factor‐β superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor‐α (TNF‐α) in models of lipopolysaccharide (LPS)‐induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum‐free conditions. Freshly isolated human neutrophils contained 20‐fold more activin A than blood mononuclear cells as measured by enzyme‐linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF‐α, but were unable to respond to LPS directly. Although TNF‐α stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF‐α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF‐α‐induced activin release, and the secretion of activin A was not due to TNF‐α‐induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF‐α may contribute to the early peak in circulating activin A levels during acute inflammation.

Downregulation of Interleukin-18-Mediated Cell Signaling and Interferon Gamma Expression by the Hepatitis B Virus e Antigen

ABSTRACT The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.