Modulation of the Immune Response by Nematode Secreted Acetylcholinesterase Revealed by Heterologous Expression in Trypanosoma musculi.

Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host.

Surfactant Protein-D Is Essential for Immunity to Helminth Infection.

Pulmonary epithelial cell responses can enhance type 2 immunity and contribute to control of nematode infections. An important epithelial product is the collectin Surfactant Protein D (SP-D). We found that SP-D concentrations increased in the lung following Nippostrongylus brasiliensis infection; this increase was dependent on key components of the type 2 immune response. We carried out loss and gain of function studies of SP-D to establish if SP-D was required for optimal immunity to the parasite. N. brasiliensis infection of SP-D-/- mice resulted in profound impairment of host innate immunity and ability to resolve infection. Raising pulmonary SP-D levels prior to infection enhanced parasite expulsion and type 2 immune responses, including increased numbers of IL-13 producing type 2 innate lymphoid cells (ILC2), elevated expression of markers of alternative activation by alveolar macrophages (alvM) and increased production of the type 2 cytokines IL-4 and IL-13. Adoptive transfer of alvM from SP-D-treated parasite infected mice into naïve recipients enhanced immunity to N. brasiliensis. Protection was associated with selective binding by the SP-D carbohydrate recognition domain (CRD) to L4 parasites to enhance their killing by alvM. These findings are the first demonstration that the collectin SP-D is an essential component of host innate immunity to helminths.

The M3 muscarinic receptor is required for optimal adaptive immunity to helminth and bacterial infection.

Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.

Attenuated Bordetella pertussis vaccine protects against respiratory syncytial virus disease via an IL-17-dependent mechanism.

RATIONALE: We attenuated virulent Bordetella pertussis by genetically eliminating or detoxifying three major toxins. This strain, named BPZE1, is being developed as a possible live nasal vaccine for the prevention of whooping cough. It is immunogenic and safe when given intranasally in adult volunteers. OBJECTIVES: Before testing in human infants, we wished to examine the potential effect of BPZE1 on a common pediatric infection (respiratory syncytial virus [RSV]) in a preclinical model. METHODS: BPZE1 was administered before or after RSV administration in adult or neonatal mice. Pathogen replication, inflammation, immune cell recruitment, and cytokine responses were measured. MEASUREMENTS AND MAIN RESULTS: BPZE1 alone did not cause overt disease, but induced efflux of neutrophils into the airway lumen and production of IL-10 and IL-17 by mucosal CD4(+) T cells. Given intranasally before RSV infection, BPZE1 markedly attenuated RSV, preventing weight loss, reducing viral load, and attenuating lung cell recruitment. Given neonatally, BPZE1 also protected against RSV-induced weight loss even through to adulthood. Furthermore, it markedly increased IL-17 production by CD4(+) T cells and natural killer cells and recruited regulatory cells and neutrophils after virus challenge. Administration of anti-IL-17 antibodies ablated the protective effect of BPZE1 on RSV disease. CONCLUSIONS: Rather than enhancing RSV disease, BPZE1 protected against viral infection, modified viral responses, and enhanced natural mucosal resistance. Prevention of RSV infection by BPZE1 seems in part to be caused by induction of IL-17. Clinical trial registered with www.clinicaltrials.gov (NCT 01188512).

Differential Regulation of Allergic Airway Inflammation by Acetylcholine.

Acetylcholine (ACh) from neuronal and non-neuronal sources plays an important role in the regulation of immune responses and is associated with the development of several disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell (ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection and therefore sought to determine whether this also plays a role in allergic inflammation. RoraCre+ChatLoxP mice (in which ILC2s cannot synthesize ACh) were exposed to an allergenic extract of the fungus Alternaria alternata, and immune responses in the airways and lung tissues were analyzed. Airway neutrophilia and expression of the neutrophil chemoattractants CXCL1 and CXCL2 were enhanced 24 h after exposure, suggesting that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic inflammation. The effect of non-selective depletion of ACh was examined by intranasal administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh in this manner resulted in a more profound enhancement of neutrophilia and chemokine expression, suggesting multiple cellular sources for the release of ACh. In contrast, depletion of ACh inhibited Alternaria-induced activation of ILC2s, suppressing the expression of IL-5, IL-13, and subsequent eosinophilia. Depletion of ACh reduced macrophages with an alternatively activated M2 phenotype and an increase in M1 macrophage marker expression. These data suggest that ACh regulates allergic airway inflammation in several ways, enhancing ILC2-driven eosinophilia but suppressing neutrophilia through reduced chemokine expression.

Characterisation of the secreted apyrase family of Heligmosomoides polygyrus.

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.

Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths.

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.

Functional analysis of effector and regulatory T cells in a parasitic nematode infection.

Parasitic nematodes typically modulate T-cell reactivity, primarily during the chronic phase of infection. We analyzed the role of CD4-positive (CD4+) T effector (T(eff)) cells and regulatory T (T(reg)) cells derived from mice chronically infected with the intestinal nematode Heligmosomoides polygyrus. Different CD4+ T-cell subsets were transferred into naïve recipients that were subsequently infected with H. polygyrus. Adoptive transfer of conventional T(eff) cells conferred protection and led to a significant decrease in the worm burdens of H. polygyrus-infected recipients. Roughly 0.2% of the CD4+ T cells were H. polygyrus specific based on expression of CD154, and cells producing interleukin 4 (IL-4) and IL-13 were highly enriched within the CD154+ population. In contrast, adoptive transfer of T(reg) cells, characterized by the markers CD25 and CD103 and the transcription factor Foxp3, had no effect on the worm burdens of recipients. Further analysis showed that soon after infection, the number of Foxp3+ T(reg) cells temporarily increased in the inflamed tissue while effector/memory-like CD103+ Foxp+ T(reg) cells systemically increased in the draining lymph nodes and spleen. In addition, T(reg) cells represented a potential source of IL-10 and reduced the expression of IL-4. Finally, under in vitro conditions, T(reg) cells from infected mice were more potent suppressors than cells derived from naïve mice. In conclusion, our data indicate that small numbers of T(eff) cells have the ability to promote host protective immune responses, even in the presence of T(reg) cells.

The Helminth-Derived Immunomodulator AvCystatin Reduces Virus Enhanced Inflammation by Induction of Regulatory IL-10+ T Cells.

Respiratory Syncytial Virus (RSV) is a major pathogen causing low respiratory tract disease (bronchiolitis), primarily in infants. Helminthic infections may alter host immune responses to both helminths and to unrelated immune triggers. For example, we have previously shown that filarial cystatin (AvCystatin/Av17) ameliorates allergic airway inflammation. However, helminthic immunomodulators have so far not been tested in virus-induced disease. We now report that AvCystatin prevents Th2-based immunopathology in vaccine-enhanced RSV lung inflammation, a murine model for bronchiolitis. AvCystatin ablated eosinophil influx, reducing both weight loss and neutrophil recruitment without impairing anti-viral immune responses. AvCystatin also protected mice from excessive inflammation following primary RSV infection, significantly reducing neutrophil influx and cytokine production in the airways. Interestingly, we found that AvCystatin induced an influx of CD4+ FoxP3+ interleukin-10-producing T cells in the airway and lungs, correlating with immunoprotection, and the corresponding cells could also be induced by adoptive transfer of AvCystatin-primed F4/80+ macrophages. Thus, AvCystatin ameliorates enhanced RSV pathology without increasing susceptibility to, or persistence of, viral infection and warrants further investigation as a possible therapy for virus-induced airway disease.

Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo.

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

Live attenuated B. pertussis BPZE1 rescues the immune functions of Respiratory Syncytial virus infected human dendritic cells by promoting Th1/Th17 responses.

Respiratory Syncytial virus (RSV) is the leading cause of acute lower respiratory tract viral infection in young children and a major cause of winter hospitalization. Bordetella pertussis is a common cause of bacterial lung disease, affecting a similar age group. Although vaccines are available for B. pertussis infection, disease rates have recently increased in many countries. We have therefore developed a novel live attenuated B. pertussis strain (BPZE1), which has recently undergone a successful clinical phase I trial. In mice, BPZE1 provides protection against disease caused by respiratory viral challenge. Here, we analyze the effect of BPZE1 on antiviral T cell responses induced by human monocyte-derived dendritic cells (MDDC). We found that BPZE1 influences antiviral immune responses at several levels, enhancing MDDC maturation, IL-12p70 production, and shifting T cell cytokine profile towards a Th1/Th17 pattern. These data were supported by the intracellular signaling analysis. RSV infection of MDDC caused MyD88-independent STAT1 phosphorylation, whereas BPZE1 activated MyD88-dependent signaling pathways; co-infection caused both pathways to be activated. These findings suggest that BPZE1 given during infancy might improve the course and outcome of viral lung disease in addition to providing specific protection against B. pertussis infection.

Natural and vaccine-mediated immunity to Salmonella Typhimurium is impaired by the helminth Nippostrongylus brasiliensis.

BACKGROUND: The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. METHODOLOGY/PRINCIPAL FINDINGS: Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. CONCLUSION: These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.

IL-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice.

Interleukin (IL-) 10 is a pleiotropic cytokine with broad immunosuppressive functions, particularly at mucosal sites such as the intestine and lung. Here we demonstrate that infection of BALB/c mice with respiratory syncytial virus (RSV) induced IL-10 production by CD4(+) and CD8(+) T cells in the airways at later time points (e.g. day 8); a proportion of these cells also co-produced IFN-γ. Furthermore, RSV infection of IL-10(-/-) mice resulted in more severe disease with enhanced weight loss, delayed recovery and greater cell infiltration of the respiratory tract without affecting viral load. In addition, IL-10(-/-) mice had a pronounced airway neutrophilia and heightened levels of pro-inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid. Notably, the proportion of lung T cells producing IFN-γ was enhanced, suggesting that IL-10 may act in an autocrine manner to dampen effector T cell responses. Similar findings were made in mice treated with anti-IL-10R antibody and infected with RSV. Therefore, IL-10 inhibits disease and inflammation in mice infected with RSV, especially during recovery from infection.

A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages.

The coincidence between infections with parasitic worms and the reduced prevalence of allergic disease in humans and in animal models has prompted the search for helminth molecules with antiallergic and antiinflammatory potential. We report herein that filarial cystatin, a secreted protease inhibitor of filarial nematodes, suppresses Th2-related inflammation and the ensuing asthmatic disease in a murine model of OVA-induced allergic airway responsiveness. Treatment with recombinant filarial cystatin inhibited eosinophil recruitment, reduced levels of OVA-specific and total IgE, down-regulated IL-4 production, and suppressed allergic airway hyperreactivity when applied during or after sensitization and before challenge with the allergen. Depletion of macrophages by clodronate-containing liposomes prevented the curative effects and restored the levels of infiltrating cells, IgE, and allergic airway reactivity. Blocking of IL-10 by application of anti-IL-10 receptor Abs restored the reduced number of infiltrating cells and the levels of OVA-specific IgE. In contrast, depletion of regulatory T cells by anti-CD25 Abs had only limited effects. Cystatin also modulated macrophage-mediated inflammation in a murine model of dextran sulfate sodium-induced colitis, leading to reduction of inflammatory infiltrations and epithelial damage. Our data demonstrate that treatment with a single helminth protein can exert the antiallergic effects of helminth infections.