Donald A. Harn

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 α1–3-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.

Lacto-N-fucopentaose III found on Schistosoma mansoni egg antigens functions as adjuvant for proteins by inducing Th2-type response.

We have recently demonstrated that induction of Th2 responses by Schistosoma mansoni egg Ag is largely due to carbohydrates on the Ag functioning as adjuvants. Lacto-N-fucopentaose III (LNFPIII), a polylactosamine sugar, is the predominant carbohydrate found in S. mansoni egg Ag. Therefore, using neoglycoprotein, we investigated whether LNFPIII induces in vivo Th2 response and functions as an adjuvant. Following intranasal immunization with LNFPIII linked to human serum albumin (HSA) (HSA-LNFPIII), BALB/c mice mounted a strong Th2 response and produced significantly higher levels of total IgE as well as HSA-specific IgG, IgG1, and IgE. HSA-LNFPIII was over 1000-fold more potent in inducing Ab production as compared with HSA alone. Although LNFPIII itself did not function as an epitope for either IgG or IgE, its conjugation with protein was essential for the adjuvant activity. Moreover, fucose residue on LNFPIII was crucial for induction of Ab production. Nasal lymphocytes from mice immunized with HSA-LNFPIII produced IL-4, IL-5, and IL-10, but not IFN-γ following in vitro stimulation with HSA or HSA-LNFPIII. In addition, these activated nasal lymphocytes also showed a significant increase of B7-2 expression on B220-positive cells. Furthermore, not only intranasal but also both i.p. and s.c. immunization with HSA-LNFPIII induced significant production of HSA-specific Abs compared with the immunization with HSA alone, suggesting that the activity of LNFPIII was not restricted on particular route of immunization. These results demonstrate that Lewis type carbohydrate LNFPIII can function as an adjuvant by their ability to induce a Th2 response.

Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference

In this paper, we used the genetic manipulation technique known as RNA-interference to suppress the expression of a target, cathepsin B, gene in the platyhelminth parasite, Schistosoma mansoni. Parasites were cultured for 6 days in the presence of double stranded RNA derived from the cathepsin B cDNA sequence or from two control sequences. Relative to the controls, the cathepsin B double stranded RNA-treated group exhibited lower levels of cathepsin B as determined by immuno-staining and by enzyme activity measurements. Additionally, using the reverse transcriptase-PCR, suppression was seen in the inability to detect cathepsin B cDNA, using RNA obtained from those parasites. This ability to manipulate gene expression represents a powerful new tool for investigating gene function in these debilitating human parasites.

The Schistosome Oligosaccharide Lacto-N-neotetraose Expands Gr1+ Cells That Secrete Anti-inflammatory Cytokines and Inhibit Proliferation of Naive CD4+ Cells: A Potential Mechanism for Immune Polarization in Helminth Infections

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1+/CD11b+/F4/80+, as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1+/F4/80+ population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-β ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1+ cells adoptively suppressed naive CD4+ T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4+ cells involved cell contact and was dependent on IFN-γ and NO, with a discrete role played by IL-10. Coculture of naive CD4+T cells with Gr1+ suppressor cells did not lead to CD4+ T cell apoptosis, although it did imprint on naive CD4+ T cells a response characterized by lower levels of IFN-γ, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.

A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-γ and Nitric Oxide-Dependent Mechanism

Lacto-N-fucopentaose III (LNFPIII) is found in human milk and on the Th2 driving helminth parasite Schistosoma mansoni. This pentasaccharide drives Th2-type responses in vivo and in vitro when conjugated to a carrier. In an attempt to further understand early events in Th1 to Th2 switching, we examined phenotypic and functional changes in peritoneal cell populations in BALB/c and SCID mice following LNFPIII-dextran injection. We found that i.p. injection with LNFPIII-dextran resulted in rapid (<20 h) expansion of the Gr1+ subpopulation of F4/80+/CD11b+ peritoneal cells, comprising up to 75% of F4/80+/CD11b+ peritoneal cells compared with 18% in uninjected or dextran-injected mice. Functionally, these cells suppressed anti-CD3- and anti-CD28-induced proliferation of naive CD4+ T cells. LNFPIII-dextran also expanded functional Gr1+ suppressor macrophages in SCID mice, demonstrating that expansion and function of suppressor cells did not require T cells. Suppression in both BALB/c and SCID mice was NO and IFN-γ dependent, as addition of inhibitors of inducible NO synthase (NG-monomethyl-l-arginine), as well as anti-IFN-γ Abs, restored the ability of CD4+ T cells to proliferate in vitro. Depletion of the F4/80+ subset of Gr1+ cells eliminated the suppressive activity of peritoneal exudate cells showing that these cells were macrophages. Thus, LNFPIII-dextran rapidly expands the Gr1+ suppressor macrophage population in the peritoneal cavities of otherwise naive mice. These Gr1+ cells suppress proliferation of naive CD4+ T cells in an NO-dependent mechanism, and may play a regulatory role in the switching of Th1- to Th2-type responses.

Immune biasing by helminth glycans

The ability of helminth parasites to drive polarized Th2 responses has been known for some time. Interestingly, many recent studies have shown that helminth‐expressed glycan activation of host immune cells accounts for much of the anti‐inflammatory and Th2‐biasing observed. This microreview attempts to cover the biology of expression of immunomodulatory glycans in various helminth parasites, the immune cells they interact with including the production of cytokines, chemokines and antibodies. We also discuss the potential   cell   surface   receptors   which   are   capable of binding certain glycans and the known mechanisms which ultimately lead to production of anti‐inflammatory   mediators   as   well   as   polarizing   CD4+ T‐cell responses to Th2‐type in the host. Lastly, we discuss a novel mechanism for activation of antigen‐presenting cells by a specific helminth glycan that leads to maturation of Type 2 dendritic cells.

Mannan-Binding Lectin Enhances Susceptibility to Visceral Leishmaniasis

ABSTRACT Levels of the serum opsonin mannan-binding lectin (MBL) were directly correlated with the probability of developing visceral leishmaniasis. Monocytes infected with MBL-opsonized Leishmania chagasi promastigotes secreted higher levels of tumor necrosis factor alpha and interleukin-6 than cells infected with nonopsonized parasites. Our findings indicate that MBL can modulate the clinical outcome of infection with L. chagasi and the function of infected macrophages.

Human Immune Responses to Schistosoma mansoni Vaccine Candidate Antigens

ABSTRACT To determine the naturally occurring immunological responses to theSchistosoma mansoni antigens paramyosin, IrV-5, Sm-23 (MAP-3), and triose phosphate isomerase (MAP-4), a total of 119 subjects from an area of endemicity for schistosomiasis, including “resistant” subjects (n = 17) were evaluated. Specific immunoglobulin G1 (IgG1), IgG2, IgG3, IgG4, and IgA levels for each of the antigens and the cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC) were determined. Although all the subjects had a high degree of contaminated water exposure, their infection levels were variable (0 to 1,128 eggs/g of stool). There were direct correlations between infection levels and levels of SWAP- and paramyosin-specific IgG1 and IgG4 (P < 0.05). However, an inverse correlation between infection levels and specific IgG2 to IrV-5 (P < 0.01) was observed. The evaluation of the cytokine profile (interleukin 5 [IL-5], IL-10, gamma interferon [IFN-γ], and tumor necrosis factor alpha) in response to these antigens showed inverse correlations between the degree of infection and IFN-γ levels in PBMC supernatants stimulated with paramyosin (P < 0.05) and IrV-5 (P < 0.01). Additionally, inverse correlations between the degree of infection and IL-5 levels in MAP-3- and MAP-4-stimulated PBMC supernatants (P < 0.01) were found. Logistic regression analysis was performed to adjust the results of cytokine profile by age. IL-5 production in MAP-3-stimulated PBMC supernatants was associated with lower infection levels (odds ratio = 11.2 [95% confidence interval, 2.7 to 45.8]).

Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice

Schistosomes are helminth parasites infecting at least 200 million people worldwide. In this study, we evaluated the feasibility of using a nucleic acid vaccine to induce protective immune responses to the Schistosoma mansoni integral membrane protein Sm23. C57BL/6 mice were immunized by intramuscular injection in three separate vaccination trials. ELISA and Western Blot analyses indicated that mice immunized with a DNA plasmid construct encoding Sm23 (Sm23-pcDNA) generated specific IgG for Sm23, while sera from mice immunized with the control pcDNA plasmid did not. The vaccine elicited IgG(2a), and IgG(1) antibody isotypes. We also tested the adjuvant activity of IL-12 and IL-4 on humoral responses to Sm23. Co-immunization with plasmid encoding IL-12 did not affect the level of anti-Sm23 IgG(2a), but did reduce the IgG(1) level. In contrast, co-injection with a plasmid encoding IL-4 significantly reduced the level of anti-Sm23 IgG(2a), while the level of IgG(1) was largely unchanged. Importantly, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection (21-44%, P<0.001-0.02). Co-administration of plasmids encoding either IL-12 or IL-4 did not significantly enhance this protective effect.

Modulation of host immune responses by helminth glycans

Summary:  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.