Immune modulation by Lacto-N-fucopentaose III in experimental autoimmune encephalomyelitis.

Parasitic infections frequently lead to immune deviation or suppression. However, the application of specific parasitic molecules in regulating autoimmune responses remains to be explored. Here we report on the immune modulatory function of Lacto-N-fucopentaose III (LNFPIII), a schistosome glycan, in an animal model for multiple sclerosis. We found that LNFPIII treatment significantly reduced the severity of experimental autoimmune encephalomyelitis (EAE) and CNS inflammation, and skewed peripheral immune response to a Th2 dominant profile. Inflammatory monocytes (IMCs) purified from LNFPIII-treated mice had increased expression of nitric oxide synthase 2, and mediated T cell suppression. LNFPIII treatment also significantly increased mRNA expression of arginase-1, aldehyde dehydrogenase 1 subfamily A2, indoleamine 2,3-dioxygenase and heme oxygenase 1 in splenic IMCs. Furthermore, LNFPIII treatment significantly reduced trafficking of dendritic cells across brain endothelium in vitro. In summary, our study demonstrates that LNFPIII glycan treatment suppresses EAE by modulating both innate and T cell immune response.

Modulation of host immune responses by helminth glycans.

Parasitic infections regulate/alter host immune responses. Among parasitic infections, helminth infection often leads to systemic immune suppression or anergy. Helminth infection or helminth extracts drive CD4+ T-helper (Th) cell responses towards Th2 type and activate antigen-presenting cells (APCs) such that these cells express an anti-inflammatory phenotype. Among the myriad molecules present on or secreted by helminth parasites, glycans have been shown to be key in inducing Th2-type and anti-inflammatory immune responses. The majority of studies on immune modulatory helminth glycans have focused on Lacto-N-fucopentaose III and LewisX. When presented as glycol-conjugates, with multiple copies of the sugars conjugated to a carrier molecule, these compounds activate APCs, inducing an alternative activation pattern, whose phenotypic profile is substantially different than that seen using pro-inflammatory activators such as lipopolysaccharide. Though the mechanism of APC activation by LNFPIII/LewisX glycoconjugates has not been fully elucidated, it involves C-type lectin ligation on the surface of APCs, with subsequent antagonism of Toll-like receptor signaling. In this article, we discuss the APC surface receptors known to play roles in LNFPIII/LewisX induced alternative activation of APCs. We also discuss what is currently known regarding downstream signaling pathways, closing with a discussion of future research directions for this field of investigation including the potential use of immune modulatory glycans as vaccine adjuvants and anti-inflammatory therapeutics.

The immunomodulatory glycan LNFPIII initiates alternative activation of murine macrophages in vivo.

The early pathogen-macrophage interactions that help drive macrophage maturation towards classically or alternatively activated are largely unknown. To examine this question we utilized the immunomodulatory glycan Lacto-N-fucopentaose III (LNFPIII), which contains the Lewis X (LeX) trisaccharide, to activate murine peritoneal macrophages in vivo. Because LNFPIII is known to induce anti-inflammatory responses, we asked if LNFPIII stimulation of macrophages in vivo initiates alternative activation events such as upregulation of Arginase 1, Ym1, FIZZ-1, MGL-1 or macrophage mannose receptor (MMR). Examination of peritoneal exudate cells from mice 20 hr post-LNFPIII injection demonstrated increased Arginase 1 activity, at the mRNA and protein levels, coincident with undetectable inducible nitric oxide synthase expression or nitric oxide production. In addition to Arginase 1, Ym1 expression was also significantly upregulated at 20 and 48 hr after LNFPIII exposure in vivo. However, the expression of FIZZ-1, MGL-1, and MMR was not changed in these macrophages. In an attempt to determine activation requirements for functional activity, we adoptively transferred antigen-pulsed, in vivo LNFPIII activated macrophages into naïve recipients and found that they were capable of triggering recipient T cells to secrete elevated levels of interleukin (IL)-10 and IL-13 compared to mice receiving control macrophages. Together, these data demonstrate that upregulation of expression of Arginase 1 and Ym1 occur very early in activation of macrophages, and can be independent of other alternatively activated (AA) macrophage markers. Importantly, these early events appear to be IL-4/IL-13-independent in our model. In the future we hope to determine if upregulation of these initial AA maturational events is sufficient for these macrophages to exert immunoregulatory activity in vivo.

Prevention of psoriasis-like lesions development in fsn/fsn mice by helminth glycans.

The helminth glycan LNFPIII is an immunomodulatory molecule, driving CD4(+) Th2-type biasing as well as immune suppression. Psoriasis is an autoimmune disease where the immune mechanisms as well as the antigens responsible for development of immune autoreactivity are still not known. In the absence of defined immunological mechanisms, we asked whether LNFPIII would function as novel therapy for psoriasis. We tested the therapeutic efficacy of LNFPIII using the flaky skin (fsn)/fsn mutant mouse model of psoriasis-like lesion development. We found that treatment of mice with LNFPIII prevented the appearance of psoriatic skin lesions on fsn/fsn mice. Examination of the skin 2 weeks after treatment demonstrated that prevention of skin lesions was associated with maintenance of normal epidermis thickness in LNFPIII-treated mice as compared with a significantly thickened epidermis in control treated and diseased mice. In addition, cells from skin of LNFPIII-treated mice produced lower amounts of interferon-gamma as compared with cells from skin of control treated diseased mice. Examination of macrophages and T cells from peripheral lymph nodes of control and LNFPIII-treated fsn/fsn mice showed that glycan treatment reduced the numbers of Gr1(+)F4/80(+) macrophages and the numbers of CD8(+) T cells, restoring the numbers of these two cell populations as well as the CD4 : CD8 ratio to near normal levels. Overall, the results from this study suggest that the helminth immunomodulatory glycan LNFPIII functions to prevent development of psoriatic-like skin lesions in fsn/fsn mice.

LNFPIII/LeX-stimulated macrophages activate natural killer cells via CD40-CD40L interaction.

Lacto-N-fucopentaose III (LNFPIII) is a human milk sugar containing the biologically active Lewis X (LeX) trisaccharide. LNFPIII/LeX is also expressed by immunosuppressive helminth parasites, by bacteria, and on a number of tumor/cancer cells. In this report, we first demonstrate that LNFPIII activates macrophages in vitro as indicated by upregulation of Gr-1 expression on F4/80(+) cells. Further, we investigated the effect of LNFPIII-activated macrophages on NK cell activity. We found that LNFPIII-stimulated F4/80(+) cells were able to activate NK cells, inducing upregulation of CD69 expression and gamma interferon (IFN-gamma) production. The experiments show that NK cell activation is macrophage dependent, since NK cells alone did not secrete IFN-gamma in response to LNFPIII. Furthermore, we found that activation of NK cells by glycan-stimulated macrophages required cell-cell contact. As part of the cell-cell contact mechanism, we determined that CD40-CD40L interaction was critical for IFN-gamma secretion by NK cells, as the addition of anti-CD40L antibodies to the coculture blocked IFN-gamma production. We also demonstrated that LNFPIII-stimulated macrophages secrete prostaglandin E(2), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) but a very low level of IL-12. Interestingly, addition of anti-TNF-alpha, anti-IL-10, or anti-IL-12 monoclonal antibodies did not significantly alter NK cell activity. Our data show that these soluble mediators are not critical for LNFPIII-stimulated macrophage activation of NK cells and provide further evidence for the importance of cell-cell contact and CD40-CD40L interactions between macrophages and NK cells.

Comparison of results using the gel microdrop cytokine secretion assay with ELISPOT and intracellular cytokine staining assay.

The gel microdrop (GMD) secretion assay involves encapsulating single cells in a biotinylated agarose matrix, addition of a streptavidin bridge, diffusion of a biotinylated capture antibody, and detection of secreted molecules using a fluorescently labeled reporter antibody. Using flow cytometry, encapsulated cells can be analyzed or recovered based on cell type and secretory profile. Using murine Th2 cell line D10.G4.1 as a model, we recently demonstrated the feasibility of using the GMD cytokine secretion assay combined with flow cytometry to detect IL-4-producing cells after stimulation with the mitogen, Con A. In addition, subpopulations of encapsulated cells secreting IL-4 were simultaneously characterized by immunophenotyping. We found good correlation between results using the GMD cytokine secretion assay and results with the standard ELISPOT and intracellular cytokine (ICC) assays. The GMD cytokine secretion assay permits simultaneous detection of secreted cytokine and determination of cell surface phenotype on viable, single cells. Moreover, using fluorescence activated cell sorting (FACS), secreting cells of interest can be sorted, recovered, and cultured for further studies.

Maturation of dendritic cell 2 phenotype by a helminth glycan uses a Toll-like receptor 4-dependent mechanism.

The biology of pathogen-associated molecular patterns (PAMPs) stimulating APCs to differentiate into a Th1-promoting phenotype has been well characterized. Conversely, not a single pathogen product that promotes a Th2 phenotype has been rigorously identified. Strong Th2 responses and dendritic cell 2 maturation are driven by helminth extracts, and carbohydrates have been shown to be responsible for much of this activity. In this study, we show that a helminth carbohydrate, lacto-N-fucopentaose III (LNFPIII) functions as an innate Th2 promoter via its action on murine dendritic cells, with the alpha1-3-linked fucose required for this activity. In contrast to Th1-type PAMPs, which activate extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, the Th2 PAMP LNFPIII preferentially activates extracellular signal-regulated kinase. Furthermore, the ability of LNFPIII to drive DC2 maturation is dependent on signaling via Toll-like receptor 4. These data support a new understanding of how APCs integrate signaling pathways to produce a Th1- or Th2-promoting phenotype.