Patrick Secrest

A red meat-derived glycan promotes inflammation and cancer progression

Significance We present an unusual mechanism for the well-known association between red meat consumption and carcinoma risk involving the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). We first evaluate the Neu5Gc content of various foods to show that red meats are particularly rich in orally bioavailable Neu5Gc and then investigate human-like Neu5Gc-deficient mice fed this form of Neu5Gc. When such mice were challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Long-term exposure to this combination resulted in a significantly higher incidence of carcinomas (five-fold increase) and an association with Neu5Gc accumulation in the tumors. Similar mechanisms may contribute to the association of red meat consumption with other diseases, such as atherosclerosis and type 2 diabetes, which are also exacerbated by inflammation. A well known, epidemiologically reproducible risk factor for human carcinomas is the long-term consumption of “red meat” of mammalian origin. Although multiple theories have attempted to explain this human-specific association, none have been conclusively proven. We used an improved method to survey common foods for free and glycosidically bound forms of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc), showing that it is highly and selectively enriched in red meat. The bound form of Neu5Gc is bioavailable, undergoing metabolic incorporation into human tissues, despite being a foreign antigen. Interactions of this antigen with circulating anti-Neu5Gc antibodies could potentially incite inflammation. Indeed, when human-like Neu5Gc-deficient mice were fed bioavailable Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Such mice are already prone to develop occasional tumors of the liver, an organ that can incorporate dietary Neu5Gc. Neu5Gc-deficient mice immunized against Neu5Gc and fed bioavailable Neu5Gc developed a much higher incidence of hepatocellular carcinomas, with evidence of Neu5Gc accumulation. Taken together, our data provide an unusual mechanistic explanation for the epidemiological association between red meat consumption and carcinoma risk. This mechanism might also contribute to other chronic inflammatory processes epidemiologically associated with red meat consumption.

Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer

Significance In vitro and in vivo data indicate that hypersialylated tumor cells can engage Siglec-9 on myelomonocytic cells and influence the outcome of the interaction, depending on the stage of tumor growth and the microenvironment. On one hand, engagement of Siglec-9 or Siglec-E by tumor-associated ligands inhibited immunosurveillance and tumor cell killing during establishment of autologous tumors and new metastatic foci. On the other hand, inhibition of tumor-associated macrophages through Siglec-9 led to M1 polarization and reduced growth-promoting inflammation within the tumor microenvironment. This demonstrates a previously unidentified dualistic function of Siglec-9 during cancer progression. A functional polymorphism of Siglec-9 correlated with altered survival of lung cancer patients, suggesting that Siglec-9 might be therapeutically targeted. Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9–expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E–deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-E–deficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E–deficient mice. In keeping with this, Siglec-E–deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in non–small-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.

Sexual selection by female immunity against paternal antigens can fix loss of function alleles

Humans lack the common mammalian cell surface molecule N-glycolylneuraminic acid (Neu5Gc) due to a CMAH gene inactivation, which occurred approximately three million years ago. Modern humans produce antibodies specific for Neu5Gc. We hypothesized that anti-Neu5Gc antibodies could enter the female reproductive tract and target Neu5Gc-positive sperm or fetal tissues, reducing reproductive compatibility. Indeed, female mice with a human-like Cmah(−/−) mutation and immunized to express anti-Neu5Gc antibodies show lower fertility with Neu5Gc-positive males, due to prezygotic incompatibilities. Human anti-Neu5Gc antibodies are also capable of targeting paternally derived antigens and mediate cytotoxicity against Neu5Gc-bearing chimpanzee sperm in vitro. Models of populations polymorphic for such antigens show that reproductive incompatibility by female immunity can drive loss-of-function alleles to fixation from moderate initial frequencies. Initially, the loss of a cell-surface antigen can occur due to drift in isolated populations or when natural selection favors the loss of a receptor exploited by pathogens, subsequently the same loss-of-function allele can come under sexual selection because it avoids being targeted by the female immune system. Thus, we provide evidence of a link between sexual selection and immune function: Antigenicity in females can select against foreign paternal antigens on sperm and rapidly fix loss-of-function alleles. Similar circumstances existed when the CMAH null allele was polymorphic in ancestral hominins, just before the divergence of Homo from australopithecines.

Sialidases on mammalian sperm mediate deciduous sialylation during capacitation

Background: Mammalian sperm lose sialic acids during capacitation through unknown mechanisms. Results: Sialidases Neu1 and Neu3 are present on sperm. Their activity is required for capacitation and zona pellucida binding. Conclusion: Sperm sialidases modulate sperm surface sialic acids en route to fertilization. Significance: Understanding the mechanism of deciduous sialylation in sperm provides novel insights into sperm function and glycan-mediated fertility. Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

Inverse hormesis of cancer growth mediated by narrow ranges of tumor-directed antibodies

Significance We have previously shown that antibodies directed against tumor-associated antigens containing the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc) can influence tumor progression. Here, we analyzed growth of Neu5Gc-positive tumors in Neu5Gc-deficient mice, following administration of increasing concentrations of anti-Neu5Gc antibodies. Although lower antibody concentrations stimulated tumor growth, higher concentrations inhibited growth, over a surprisingly narrow dose range. This biphasic narrow-range tumor growth response to tumor-directed antibodies (inverse hormesis) was reproduced in multiple mouse models, including one using a clinically approved monoclonal antibody. Our results are a novel experimental demonstration of how the levels of circulating antibodies can differentially influence tumor growth. These findings might have important implications in natural cancer progression and/or in cancer immunotherapy with antibodies. Compelling evidence for naturally occurring immunosurveillance against malignancies informs and justifies some current approaches toward cancer immunotherapy. However, some types of immune reactions have also been shown to facilitate tumor progression. For example, our previous studies showed that although experimental tumor growth is enhanced by low levels of circulating antibodies directed against the nonhuman sialic acid N-glycolyl-neuraminic acid (Neu5Gc), which accumulates in human tumors, growth could be inhibited by anti-Neu5Gc antibodies from a different source, in a different model. However, it remains generally unclear whether the immune responses that mediate cancer immunosurveillance vs. those responsible for inflammatory facilitation are qualitatively and/or quantitatively distinct. Here, we address this question using multiple murine tumor growth models in which polyclonal antibodies against tumor antigens, such as Neu5Gc, can alter tumor progression. We found that although growth was stimulated at low antibody doses, it was inhibited by high doses, over a linear and remarkably narrow range, defining an immune response curve (IRC; i.e., inverse hormesis). Moreover, modulation of immune responses against the tumor by altering antibody avidity or by enhancing innate immunity shifted the IRC in the appropriate direction. Thus, the dualistic role of immunosurveillance vs. inflammation in modulating tumor progression can be quantitatively distinguished in multiple model systems, and can occur over a remarkably narrow range. Similar findings were made in a human tumor xenograft model using a narrow range of doses of a monoclonal antibody currently in clinical use. These findings may have implications for the etiology, prevention, and treatment of cancer.

Siglec receptors impact mammalian lifespan by modulating oxidative stress.

Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan. DOI: http://dx.doi.org/10.7554/eLife.06184.001

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo

ABSTRACT Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1 HEP mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1 HEP mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1 HEP mice. FX remained essential for Ad5 transduction in vivo in Ext1 HEP mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.

A mouse model for dietary xenosialitis: antibodies to xenoglycan can reduce fertility

Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into reproductive tissues and secretions. Most humans also have circulating antibodies specific for this dietary xenoglycan. The potential for inflammation induced by incorporated Neu5Gc and circulating anti-Neu5Gc antibodies, termed xenosialitis, has been discussed as a factor influencing several human diseases. Potential effects of xenosialitis on human fertility remain unknown. Here, we investigate possible adverse effects of the presence of Neu5Gc on sperm or endometrium combined with anti-Neu5Gc antibodies in semen or uterine secretions in a mouse model. We use Cmah−/− mice, humanized for Neu5Gc deficiency. We find that the viability, migration, and capacitation of sperm with incorporated Neu5Gc are negatively affected when these are exposed to anti-Neu5Gc antibodies. In addition, we find that after copulation, activated uterine neutrophils and macrophages show increased phagocytosis of sperm in the presence of anti-Neu5Gc antibodies via the complement receptor 3 (C3R) and Fcγ I/II/III (Fc receptor). Furthermore, Neu5Gc in endometrial cells combined with the presence of anti-Neu5Gc antibodies alters the receptivity and decidualization of endometrial explants. These studies provide mechanistic insights on how Neu5Gc on sperm and/or endometrium combined with anti-Neu5Gc antibodies in semen and uterine fluid might contribute to unexplained human infertility.

Abstract LB-156: A diet-derived sialic acid promotes inflammation and hepatocellular cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA N-glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid derived from the diet (primarily red meat) that can be metabolically incorporated into human tissues despite being an antigen. Since this incorporation occurs in the face of circulating anti-Neu5Gc antibodies, we hypothesized that this generates an antibody-antigen reaction and leads to inflammation termed “xenosialitis”. Although red meat consumption is prominently associated with cancer development, pathological mechanisms behind this link are yet to be conclusively proven. The purpose of our research is to investigate the relationship between “xenosialitis”, inflammation and cancer. We used a Cmah null mouse model (mimicking the human lack of Neu5Gc) to provide evidence for such inflammation and cancer promotion. Anti-Neu5Gc antibodies were generated by immunizing Cmah null mice, with chimpanzee red blood cell membranes, or with human red blood cell membranes as controls. Pooled immune or control sera were repeatedly adsorbed with human red blood cells, and the resulting polyclonal antisera passively transferred into additional Cmah null mice that were on a diet with or without Neu5Gc. Mice fed with Neu5Gc and injected with anti-Neu5Gc antibodies showed dose-dependent elevation in acute phase proteins (Serum Amyloid A and Haptoglobin) in the serum as compared to controls. Such C57BL/6 mice are prone to developing hepatic adenomas and occasional carcinomas. Cmah null but not wild-type C57BL/6 mice immunized with chimpanzee red blood cell membranes and fed with Neu5Gc developed a higher frequency of hepatocellular cancer and liver histology showed Neu5Gc accumulation and foci of inflammation. In conclusion, Neu5Gc that has metabolically incorporated into tissues from the diet interacts with anti-Neu5Gc antibodies, generating xenosialitis. Potential correlations of human anti-Neu5Gc antibody levels with red meat consumption and cancer risk are being investigated. Methods to flush out the xenogenic Neu5Gc are being pursued. Similar mechanisms may be operative in other chronic inflammatory processes in diseases epidemiologically associated with red meat consumption, such as atherosclerosis. Citation Format: Annie N. Samraj, Heinz Laubli, Oliver Pearce, Patrick Secrest, Andrea E. Garcia-Bingman, Nissi Varki, Ajit Varki. A diet-derived sialic acid promotes inflammation and hepatocellular cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-156. doi:10.1158/1538-7445.AM2014-LB-156

Abstract 3659: Engagement of myelomonocytic siglecs by tumor-associated ligands modulates innate immune responses to cancer

Sialic acids are dominant glycans on vertebrate cell surfaces, mediating roles as diverse as influenza infection, leukocyte trafficking and neural plasticity. It has long been known that malignant cells upregulate sialic acids, but few reasons for this have so far been elucidated. In apparently unrelated work, we have recently found that certain pathogenic bacteria coat themselves with sialic acids, thereby dampening innate immune responses, via engagement of inhibitory Siglecs (sialic acid binding Ig-like lectins). We therefore hypothesized that hypersialylated carcinoma cells might similarly engage Siglecs and modulate immune cell function. Confirming this hypothesis, we now have the first demonstration of the involvement of Siglecs in cancer progression in vivo. Siglec-9 is the most abundant inhibitory Siglec on human neutrophils and monocytes/macrophages. We first demonstrate that ligands for Siglec-9 are strongly upregulated in human carcinomas. We further provide evidence that ligands on carcinoma cells can inhibit neutrophil activation and killing of tumor cells by neutrophils. We then show that mice lacking Siglec-E (the murine functional equivalent of Siglec-9) have an increased immunosurveillance in autochthonous and transplantation models, which could be reversed by transgenic expression of Siglec-9 in myelomonocytic cells. Studies of later phases of tumor progression however showed that Siglec-E/-9 inhibits polarization of M2 macrophages and therefore impairs angiogenesis and tumor growth. In keeping with this dualistic role of myelomonocytic Siglecs during different phases of cancer progression, survival of non-small cell lung cancer patients with a polymorphism that reduced ligand binding to Siglec-9 had initially an improved survival, suggesting an increased immunosurveillance, but this effect was lost during longer followup. Our results identify inhibitory CD33-related myelomonocytic Siglecs as important players in cancer biology and as potential targets for immunomodulatory therapy. We show that hypersialylated carcinoma cells modulate the innate immune response by engaging Siglec-9 and inhibit either immunosurveillance or cancer-related inflammation. Our data also exemplify the dualistic role of innate immune cells and their receptors in cancer progression depending on the context and the microenvironment, i.e., growth control by immunosurveillance or growth support by cancer-related inflammation. Citation Format: Heinz Laubli, Oliver M. T. Pearce, Flavio Schwarz, Lingquan Deng, Michal Stanczak, Liwen Deng, Andrea Verhagen, Patrick Secrest, Chrissy Lusk, Ann G. Schwartz, Nissi Varki, Jack Bui, Ajit Varki. Engagement of myelomonocytic siglecs by tumor-associated ligands modulates innate immune responses to cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2014-3659