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Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3′ of each of the two sugars, resulting in a 3′-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl β-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3′ position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.

Activation of inflammatory caspases, such as caspase-11 and caspase-4, by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infections. This cytosolic LPS sensing pathway results in activation of a pore-forming protein, gasdermin D, which mediates an inflammatory form of cell death called pyroptosis. In addition, caspase-11-mediated cytosolic LPS sensing leads to the activation of caspase-1 and the maturation of IL-1b and IL-18. Importantly, cytosolic LPS-induced pyroptosis is associated with the release of intracellular proteins that act as alarmins or damage-associated molecular patterns (DAMPs) to modulate the inflammatory response and play an important role during infectious diseases and sepsis. However, the identity and function of DAMPs released downstream of caspase-11 activation are poorly characterized. Using a proteomic approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have discovered that galectin-1, a b-galactoside-binding lectin, is released as a consequence of cytosolic LPS sensing. By utilizing various mutant cell lines and primary cells we show that galectin-1 secretion is dependent on caspase-11 and gasdermin D, but not NLRP3 or caspase-1. Importantly, in vivo studies using galectin-1-deficient mice showed that galectin-1 acts as a DAMP to promote systemic inflammation and plays a detrimental role during septic shock. Additionally, human septic patients were found to have increased levels of galectin-1 in their serum. Taken together, we have characterized the secretion and function of a novel lectin DAMP released upon lytic cell death and provide new insights into outcomes of inflammasome activation.

Sepsis is a life-threatening disease caused by the immune system’s response to infection that results in excessive inflammation, organ damage and often death. In fact, sepsis is the leading cause of death in intensive care units and is responsible for more than 250,000 deaths every year in the USA. Activation of inflammatory caspases, such as caspase-11 by lipopolysaccharide (LPS) from Gram-negative bacteria, is a key mechanism of innate immune defense against infection. Intracellular sensing of LPS by caspase-11 results in pyroptosis, maturation of caspase-1, IL-1β and IL-18 and unconventional secretion of intracellular proteins that lack a leader sequence for conventional secretion via endoplasmic reticulum-Golgi route. These proteins can act as alarmins or damage associated molecular patterns (DAMPs) to regulate the inflammatory response and therefore play an important role during infectious diseases. However, caspase-11-dependent release of alarmins and DAMPs following noncanonical inflammasome activation is poorly characterized. Using a proteomics approach involving ProteomeLab’s PF2D fractionation system followed by mass spectrometry, we have profiled several new alarmins released in a caspase-11 dependent manner following Gram-negative bacterial infection. By employing various mutant mice and cells, we have defined the molecular and cellular mechanisms underlying the release of a candidate alarmin as a consequence of noncanonical inflammasome activation in vitro and in vivo. Importantly, our in vivo studies demonstrate a critical role for this alarmin in LPS shock. Overall, these findings provide new insights into potential biomarkers and therapeutic targets for blocking the lethal inflammation in sepsis.

Intracellular sensing of LPS by inflammatory caspases such as caspase-11 has emerged as a key mechanism of innate immune activation during Gram-negative bacterial infections. Cytosolic recognition of LPS by caspase-11 leads to inflammatory cell death or pyroptosis and maturation of IL-1 cytokines. Remarkably, the activation of inflammatory caspases also results in the extracellular release of alarmins and damage associated molecular patterns (DAMPs). These alarmins and DAMPs lack leader signals for classical secretion and are therefore released via a caspase-11 mediated unconventional secretory mechanism. Alarmins and DAMPs—structurally diverse molecules participating in various cellular processes under steady state conditions—are released rapidly upon infections and tissue damage. In the extracellular milieu, these proteins elicit inflammatory reactions by regulating pattern recognition receptor signaling and recruiting innate cells. Despite their potential roles in sepsis, the identity of alarmins and DAMPs released as a result of cytosolic LPS sensing and their contribution to sepsis are poorly understood. Addressing this knowledge gap, we employed an unbiased proteomic approach involving two-dimensional liquid phase fractionation system and mass spectrometry and identified alarmins released in response to caspase-11 activation by cytosolic LPS. In this presentation, the molecular basis of secretion of a candidate alarmin in vitro and in vivo as well as its function in murine models of host defense will be discussed.

Recently we showed that the interaction of MGL, expressed by tolerogenic dendritic cells (DCs), with GalNAc-decorated CD45 on effector cells inhibits T cell function leading to T cell death. The LacNAc binding lectin Gal1 also binds CD45 inducing apoptosis of Th1 and Th17 effector T cells. However, whether these lectins may act in concert to control effector T cell functions remain to be explored. Firstly, we incubated both lectins with Jurkat T cells and measured apoptosis by annexin V. We found higher and faster apoptosis induction compared to each lectin alone. A mechanistic analysis of this result by ImageStream® technology revealed that MGL triggers Gal1 expression that later colocalizes with CD45 in the membrane inducing high levels of receptor clustering and, in this manner, leads to augmented apoptosis. This effect is CD45 mediated and T cell specific, since Jurkat T cells devoid of this receptor or CD45-expressing carcinoma or adenocarcinoma cell lines are protected from apoptosis when incubated with MGL. Next, we isolated and activated mouse CD4+ T cells and showed that MGL1 but not MGL2 homologue induces apoptosis. However, there is no additive effect in primary CD4+ T cells. Although more work is required to fully elucidate the observed effects, these results indicate that both MGL and Gal1 may modify effector T cell function, through the interaction with specific CD45 glycoforms, potentially contributing to the homeostasis of the immune response.

Hormone replacement therapy with the progesterone analogue medroxyprogesterone acetate (MPA) is widely used in postmenopausal women for the treatment of endometrial conditions, and as a contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Our aim was to explore if MPA threats immunosurveillance to tumors by effecting myeloid-derived suppressor (MDSCs; CD11b+Gr1+) and NK cells in mammary tumor-bearing mice. We used the highly metastatic 4T1 breast tumor which does not express classical progesterone or glucocorticoid receptors. MPA did not affect primary tumor growth in 4T1-tumor bearing mice but promoted lung metastasis burden. This effect was accompanied by expansion of splenic CD11b+Gr1+ cells (mostly CD11b+Gr1high cells). Sorted CD11b+Gr1+ cells from MPA-treated tumor bearing mice showed a more pronounced inhibitory activity of NK cell degranulation in response to YAC-1 cells and IFN-γ production in response to cytokines than those isolated from untreated tumor-bearing mice. Also, MPA significantly increased the percentage of spleen NK cells in tumor-bearing mice with similar lung infiltration of CD11b+Gr1+ and NK cells as compared to untreated tumor-bearing mice. We conclude that in breast cancer-bearing hosts MPA promotes the accumulation of CD11b+Gr1+ which suppress NK-cell mediated anti-tumor activity potentially contributing to tumor progression and metastasis.

The engulfment of apoptotic leukocytes (efferocytosis) by macrophages during the resolution of inflammation is essential for homeostasis and results in macrophage reprogramming/immune-silencing. Here, we show CD11bhigh macrophages convert to CD11blow ones and stop efferocytosing apoptotic PMN after reaching an engulfment threshold in vivo. In addition, CD11blow macrophages are distinct from either M1 or M2 in their protein expression profile and display pro-resolving properties, such as diminished responses to different TLR ligands ex vivo and propensity to emigrate from resolving inflammation sites to lymphoid organs. Of interest, we found the pro-resolving lipid mediators resolvin E1 and D1, as well as the glucocorticoid dexamethasone (Dex) and the lectin galectin-1 enhance satiated-efferocytosis and consequently CD11blow macrophage conversion from their CD11bhigh counterparts. Deficiency in the atypical chemokine receptor D6 resulted in delayed satiation and reduced immune-silencing of macrophages, and inhibits their departure from resolving inflammation sites. In sum, satiated-efferocytosis is a novel phagocyte property of CD11blow macrophages that is regulated by pro-resolving mediators. Moreover, satiated-efferocytosis is required for CD11blow macrophage emigration from resolving inflammation sites and the return of tissue homeostasis. Thus, satiated-efferocytosis is essential for the completion of timely- and spatially-coordinated resolution of acute inflammation.

Abstract 51 Galectin-1 (Gal1) is a member of a highly conserved family of carbohydrate-binding proteins that modulate innate and adaptive immune responses and foster tumor immune escape. Through the selective recognition of specific cell-surface glycans (Gal-beta1-4-NAcGlc [N-acetyllactosamine] units on the branches of N- or O-linked glycans), Gal1 induces the apoptosis of Th1 and cytotoxic T cells. Th2 cells have different patterns of sialylation of cell-surface glycoproteins, lack Gal1 ligands, and resist Gal1-induced cell death. Regulatory T (Treg) cells are also resistant to Gal1-mediated apoptosis. In addition, Gal1 promotes hypoxia-driven tumor angiogenesis. Primary Hodgkin lymphomas (HLs) include small numbers of malignant Reed-Sternberg cells within a Th2/Treg-skewed inflammatory infiltrate. In previous studies, we found that Reed-Sternberg cells overexpress Gal1, which selectively kills Th1 and cytotoxic T cells and promotes the immunosuppressive Th2/Treg-predominant HL microenvironment. In HLs, which exhibit constitutive AP-1 activation, Gal1 overexpression is driven in large part by an AP-1-dependent enhancer. Given the immunosuppressive function of Gal1, we reasoned that the secreted protein might be a potent marker of disease activity and a novel therapeutic target. We previously developed a panel of Gal1 monoclonal antibodies and demonstrated the utility of Gal1 as a diagnostic marker of AP-1-dependent lymphoid malignancies. A potent neutralizing Gal1 antibody also protected Th1 and cytotoxic T cells from Gal1-induced apoptosis, abrogated Gal1-associated tumor angiogenesis and limited the growth of Gal1+ tumors in vivo. We have now developed a sandwich ELISA to assess the utility of Gal1 as a serum marker of disease burden in HL. After establishing the sandwich ELISA with purified recombinant Gal1 and the newly developed Gal1 antibodies, we assessed the levels of serum Gal1 in 15 healthy normal donors and 315 newly diagnosed, previously untreated HL patients from the German Hodgkin Study Group. The HL patients were enrolled on 3 tailored, risk-adapted clinical trials: HD13 for early-stage disease (clinical stage [CS] IA-IIB) with no risk factors, 94 pts); HD 14 for early stage disease with risk factors (CS I-IIA with large mediastinal mass, extranodal disease, elevated ESR or > 3 nodal areas and CS IIB with elevated ESR or > 3 nodal areas, 90 pts); and HD18 for bulky localized or advanced stage disease (CS IIB with bulky mediastinal involvement and/or extranodal involvement and CS III or IV, 131 pts). Serum Gal1 levels were significantly elevated in HL patients in comparison to normal controls (median value 2.3 X higher, p We next evaluated the potential association of Gal1 serum levels with Ann Arbor stage and B-symptoms, two major determinants for assigning HL pts to risk-adapted therapy. Gal1 levels were significantly higher in HL pts with advanced-stage disease (stage I/II vs. III/IV, p We also assessed the potential association of Gal1 levels with individual clinical prognostic factors in the Hodgkin Lymphoma International Prognostic Score (IPS). In univariate analyses, Gal1 levels were highly correlated with the following clinical risk factors: extranodal involvement (p =.01); > 3 lymph node areas (p =.0001); elevated erythrocyte sedimentation rate (ESR) (p =.0015); albumin < 4 g/dl (p =.035); hemoglobin < 10.5 g/dl (p =.035); lymphoma count < 600 per mm or < 8% of white cell count (p =.015). Consistent with these observations, HL patients with an IPS score of >= 2 had significantly higher Gal1 levels than patients with an IPS of 0 or 1 (p =.007). Direct analyses of the association between Gal1 levels and outcome await completion of the ongoing HD18 clinical trial. In conclusion, Gal1 serum levels are significantly associated with tumor burden and additional adverse clinical characteristics in newly diagnosed HL patients. Given the demonstrated role of Gal1 in tumor immune evasion and angiogenesis, analyses of circulating Gal1 levels may inform risk-adapted and targeted treatment strategies for HL patients. Disclosures: No relevant conflicts of interest to declare.