HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Malat1 Suppresses Immunity to Infection through Promoting Expression of Maf and IL-10 in Th Cells.

Despite extensive mapping of long noncoding RNAs in immune cells, their function in vivo remains poorly understood. In this study, we identify over 100 long noncoding RNAs that are differentially expressed within 24 h of Th1 cell activation. Among those, we show that suppression of Malat1 is a hallmark of CD4+ T cell activation, but its complete deletion results in more potent immune responses to infection. This is because Malat1-/- Th1 and Th2 cells express lower levels of the immunosuppressive cytokine IL-10. In vivo, the reduced CD4+ T cell IL-10 expression in Malat1-/- mice underpins enhanced immunity and pathogen clearance in experimental visceral leishmaniasis (Leishmania donovani) but more severe disease in a model of malaria (Plasmodium chabaudi chabaudi AS). Mechanistically, Malat1 regulates IL-10 through enhancing expression of Maf, a key transcriptional regulator of IL-10 Maf expression correlates with Malat1 in single Ag-specific Th cells from P. chabaudi chabaudi AS-infected mice and is downregulated in Malat1-/- Th1 and Th2 cells. The Malat1 RNA is responsible for these effects, as antisense oligonucleotide-mediated inhibition of Malat1 also suppresses Maf and IL-10 levels. Our results reveal that through promoting expression of the Maf/IL-10 axis in effector Th cells, Malat1 is a nonredundant regulator of mammalian immunity.

miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity.

Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR-132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4+ T cell function is poorly understood. Here, we show that CD4+ T cells express high levels of miR-132 and that T cell activation leads to miR-132 up-regulation. The transcriptomic hallmark of splenic CD4+ T cells lacking the miR-132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR-132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation of RP transcription. Following infection with Leishmania donovani, miR-132-/- CD4+ T cells display enhanced expression of IL-10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL-10 expression in miR-132-/- Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR-132/212-mediated regulation of RP expression is critical for optimal CD4+ T cell activation and protective immunity against pathogens.

Macrophage origin limits functional plasticity in helminth-bacterial co-infection.

Rapid reprogramming of the macrophage activation phenotype is considered important in the defense against consecutive infection with diverse infectious agents. However, in the setting of persistent, chronic infection the functional importance of macrophage-intrinsic adaptation to changing environments vs. recruitment of new macrophages remains unclear. Here we show that resident peritoneal macrophages expanded by infection with the nematode Heligmosomoides polygyrus bakeri altered their activation phenotype in response to infection with Salmonella enterica ser. Typhimurium in vitro and in vivo. The nematode-expanded resident F4/80high macrophages efficiently upregulated bacterial induced effector molecules (e.g. MHC-II, NOS2) similarly to newly recruited monocyte-derived macrophages. Nonetheless, recruitment of blood monocyte-derived macrophages to Salmonella infection occurred with equal magnitude in co-infected animals and caused displacement of the nematode-expanded, tissue resident-derived macrophages from the peritoneal cavity. Global gene expression analysis revealed that although nematode-expanded resident F4/80high macrophages made an anti-bacterial response, this was muted as compared to newly recruited F4/80low macrophages. However, the F4/80high macrophages adopted unique functional characteristics that included enhanced neutrophil-stimulating chemokine production. Thus, our data provide important evidence that plastic adaptation of MΦ activation does occur in vivo, but that cellular plasticity is outweighed by functional capabilities specific to the tissue origin of the cell.

Chronic Gastrointestinal Nematode Infection Mutes Immune Responses to Mycobacterial Infection Distal to the Gut.

Helminth infections have been suggested to impair the development and outcome of Th1 responses to vaccines and intracellular microorganisms. However, there are limited data regarding the ability of intestinal nematodes to modulate Th1 responses at sites distal to the gut. In this study, we have investigated the effect of the intestinal nematode Heligmosomoides polygyrus bakeri on Th1 responses to Mycobacterium bovis bacillus Calmette-Guérin (BCG). We found that H. polygyrus infection localized to the gut can mute BCG-specific CD4(+) T cell priming in both the spleen and skin-draining lymph nodes. Furthermore, H. polygyrus infection reduced the magnitude of delayed-type hypersensitivity (DTH) to PPD in the skin. Consequently, H. polygyrus-infected mice challenged with BCG had a higher mycobacterial load in the liver compared with worm-free mice. The excretory-secretory product from H. polygyrus (HES) was found to dampen IFN-γ production by mycobacteria-specific CD4(+) T cells. This inhibition was dependent on the TGF-ßR signaling activity of HES, suggesting that TGF-ß signaling plays a role in the impaired Th1 responses observed coinfection with worms. Similar to results with mycobacteria, H. polygyrus-infected mice displayed an increase in skin parasite load upon secondary infection with Leishmania major as well as a reduction in DTH responses to Leishmania Ag. We show that a nematode confined to the gut can mute T cell responses to mycobacteria and impair control of secondary infections distal to the gut. The ability of intestinal helminths to reduce DTH responses may have clinical implications for the use of skin test-based diagnosis of microbial infections.

S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells.

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.

Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo.

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.

MyD88 signaling inhibits protective immunity to the gastrointestinal helminth parasite Heligmosomoides polygyrus.

Helminth parasites remain one of the most common causes of infections worldwide, yet little is still known about the immune signaling pathways that control their expulsion. C57BL/6 mice are chronically susceptible to infection with the gastrointestinal helminth parasite Heligmosomoides polygyrus. In this article, we report that C57BL/6 mice lacking the adapter protein MyD88, which mediates signaling by TLRs and IL-1 family members, showed enhanced immunity to H. polygyrus infection. Alongside increased parasite expulsion, MyD88-deficient mice showed heightened IL-4 and IL-17A production from mesenteric lymph node CD4(+) cells. In addition, MyD88(-/-) mice developed substantial numbers of intestinal granulomas around the site of infection, which were not seen in MyD88-sufficient C57BL/6 mice, nor when signaling through the adapter protein TRIF (TIR domain-containing adapter-inducing IFN-ß adapter protein) was also ablated. Mice deficient solely in TLR2, TLR4, TLR5, or TLR9 did not show enhanced parasite expulsion, suggesting that these TLRs signal redundantly to maintain H. polygyrus susceptibility in wild-type mice. To further investigate signaling pathways that are MyD88 dependent, we infected IL-1R1(-/-) mice with H. polygyrus. This genotype displayed heightened granuloma numbers compared with wild-type mice, but without increased parasite expulsion. Thus, the IL-1R-MyD88 pathway is implicated in inhibiting granuloma formation; however, protective immunity in MyD88-deficient mice appears to be granuloma independent. Like IL-1R1(-/-) and MyD88(-/-) mice, animals lacking signaling through the type 1 IFN receptor (i.e., IFNAR1(-/-)) also developed intestinal granulomas. Hence, IL-1R1, MyD88, and type 1 IFN receptor signaling may provide pathways to impede granuloma formation in vivo, but additional MyD88-mediated signals are associated with inhibition of protective immunity in susceptible C57BL/6 mice.

Secretion of protective antigens by tissue-stage nematode larvae revealed by proteomic analysis and vaccination-induced sterile immunity.

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.

Antibodies and IL-3 support helminth-induced basophil expansion.

Basophils are powerful mediators of Th2 immunity and are present in increased numbers during allergic inflammation and helminth infection. Despite their ability to potentiate Th2 immunity the mechanisms regulating basophil development remain largely unknown. We have found a unique role for isotype-switched antibodies in promoting helminth-induced basophil production following infection of mice with Heligmosomoides polygyrus bakeri or Nippostrongylus brasiliensis. H. polygyrus bakeri-induced basophil expansion was found to occur within the bone marrow, and to a lesser extent the spleen, and was IL-3 dependent. IL-3 was largely produced by CD4(+)CD49b(+)NK1.1(-) effector T cells at these sites, and required the IL-4Rα chain. However, antibody-deficient mice exhibited defective basophil mobilization despite intact T-cell IL-3 production, and supplementation of mice with immune serum could promote basophilia independently of required IL-4Rα signaling. Helminth-induced eosinophilia was not affected by the deficiency in isotype-switched antibodies, suggesting a direct effect on basophils rather than through priming of Th2 responses. Although normal type 2 immunity occurred in the basopenic mice following primary infection with H. polygyrus bakeri, parasite rejection following challenge infection was impaired. These data reveal a role for isotype-switched antibodies in promoting basophil expansion and effector function following helminth infection.

Innate and adaptive type 2 immune cell responses in genetically controlled resistance to intestinal helminth infection.

The nematode Heligmosomoides polygyrus is an excellent model for intestinal helminth parasitism. Infection in mice persists for varying lengths of time in different inbred strains, with CBA and C57BL/6 mice being fully susceptible, BALB/c partially so and SJL able to expel worms within 2-3 weeks of infection. We find that resistance correlates not only with the adaptive Th2 response, including IL-10 but with activation of innate lymphoid cell and macrophage populations. In addition, the titer and specificity range of the serum antibody response is maximal in resistant mice. In susceptible strains, Th2 responses were found to be counterbalanced by IFN-γ-producing CD4(+) and CD8(+) cells, but these are not solely responsible for susceptibility as mice deficient in either CD8(+) T cells or IFN-γ remain unable to expel the parasites. Foxp3(+) Treg numbers were comparable in all strains, but in the most resistant SJL strain, this population does not upregulate CD103 in infection, and in the lamina propria the frequency of Foxp3(+)CD103(+) T cells is significantly lower than in susceptible mice. The more resistant SJL and BALB/c mice develop macrophage-rich IL-4Rα-dependent Type 2 granulomas around intestinal sites of larval invasion, and expression of alternative activation markers Arginase-1, Ch3L3 (Ym1) and RELM-α within the intestine and the peritoneal lavage was also strongly correlated with helminth elimination in these strains. Clodronate depletion of phagocytic cells compromises resistance of BALB/c mice and slows expulsion in the SJL strain. Thus, Type 2 immunity involves IL-4Rα-dependent innate cells including but not limited to a phagocyte population, the latter likely involving the action of specific antibodies.

Heligmosomoides polygyrus elicits a dominant nonprotective antibody response directed against restricted glycan and peptide epitopes.

Heligmosomoides polygyrus is a widely used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary Ab response as a subset of the secreted products in H. polygyrus excretory-secretory (HES) Ag. An immunodominant epitope is an O-linked glycan (named glycan A) carried on three highly expressed HES glycoproteins (venom allergen Ancylostoma-secreted protein-like [VAL]-1, -2, and -5), which stimulates only IgM Abs, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (glycan B), present on both a non-protein high molecular mass component and a 65-kDa molecule, is widely distributed in adult somatic tissues. Whereas the high molecular mass component and 65-kDa molecules bear phosphorylcholine, the glycan B epitope itself is not phosphorylcholine. Class-switched IgG1 Abs are found to glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary Ab responses include the same specificities while also recognizing VAL-7. Although vaccination with HES conferred complete protection against challenge H. polygyrus infection, mAbs raised against each of the glycan epitopes and against VAL-1, VAL-2, and VAL-4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective Abs against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity.

Immune modulation and modulators in Heligmosomoides polygyrus infection.

The intestinal nematode parasite Heligmosomoides polygyrus bakeri exerts widespread immunomodulatory effects on both the innate and adaptive immune system of the host. Infected mice adopt an immunoregulated phenotype, with abated allergic and autoimmune reactions. At the cellular level, infection is accompanied by expanded regulatory T cell populations, skewed dendritic cell and macrophage phenotypes, B cell hyperstimulation and multiple localised changes within the intestinal environment. In most mouse strains, these act to block protective Th2 immunity. The molecular basis of parasite interactions with the host immune system centres upon secreted products termed HES (H. polygyrus excretory-secretory antigen), which include a TGF-ß-like ligand that induces de novo regulatory T cells, factors that modify innate inflammatory responses, and molecules that block allergy in vivo. Proteomic and transcriptomic definition of parasite proteins, combined with biochemical identification of immunogenic molecules in resistant mice, will provide new candidate immunomodulators and vaccine antigens for future research.

Corrigendum: CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

[This corrects the article DOI: 10.3389/fimmu.2022.903796.].

CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Bone marrow remodeling supports hematopoiesis in response to immune thrombocytopenia progression in mice.

Immune thrombocytopenia (ITP) is an acquired autoimmune condition characterized by both reduced platelet production and the destruction of functionally normal platelets by sustained attack from the immune system. However, the effect of prolonged ITP on the more immature hematopoietic progenitors remains an open area of investigation. By using a murine in vivo model of extended ITP, we revealed that ITP progression drives considerable progenitor expansion and bone marrow (BM) remodeling. Single-cell assays using Lin-Sca1+c-Kit+CD48-CD150+ long-term hematopoietic stem cells (LT-HSCs) revealed elevated LT-HSC activation and proliferation in vitro. However, the increased activation did not come at the expense of LT-HSC functionality as measured by in vivo serial transplantations. ITP progression was associated with considerable BM vasodilation and angiogenesis, as well as a twofold increase in the local production of CXCL12, a cytokine essential for LT-HSC function and BM homing expressed at high levels by LepR+ BM stromal cells. This was associated with a 1.5-fold increase in LepR+ BM stromal cells and a 5.5-fold improvement in progenitor homing to the BM. The increase in stromal cells was transient and reverted back to baseline after platelet count returned to normal, but the vasculature changes in the BM persisted. Together, our data demonstrate that LT-HSCs expand in response to ITP and that LT-HSC functionality during sustained hematopoietic stress is maintained through an adapting BM microenvironment.

Macrophage Migration Inhibitory Factor (MIF) Is Essential for Type 2 Effector Cell Immunity to an Intestinal Helminth Parasite.

Immunity to intestinal helminths is known to require both innate and adaptive components of the immune system activated along the Type 2 IL-4R/STAT6-dependent pathway. We have found that macrophage migration inhibitory factor (MIF) is essential for the development of effective immunity to the intestinal helminth Heligmosomoides polygyrus, even following vaccination which induces sterile immunity in wild-type mice. A chemical inhibitor of MIF, 4-IPP, was similarly found to compromise anti-parasite immunity. Cellular analyses found that the adaptive arm of the immune response, including IgG1 antibody responses and Th2-derived cytokines, was intact and that Foxp3+ T regulatory cell responses were unaltered in the absence of MIF. However, MIF was found to be an essential cytokine for innate cells, with ablated eosinophilia and ILC2 responses, and delayed recruitment and activation of macrophages to the M2 phenotype (expressing Arginase 1, Chil3, and RELM-α) upon infection of MIF-deficient mice; a macrophage deficit was also seen in wild-type BALB/c mice exposed to 4-IPP. Gene expression analysis of intestinal and lymph node tissues from MIF-deficient and -sufficient infected mice indicated significantly reduced levels of Arl2bp, encoding a factor involved in nuclear localization of STAT3. We further found that STAT3-deficient macrophages expressed less Arginase-1, and that mice lacking STAT3 in the myeloid compartment (LysMCrexSTAT3fl/fl) were unable to reject a secondary infection with H. polygyrus. We thus conclude that in the context of a Type 2 infection, MIF plays a critical role in polarizing macrophages into the protective alternatively-activated phenotype, and that STAT3 signaling may make a previously unrecognized contribution to immunity to helminths.

The secretome of the filarial parasite, Brugia malayi: proteomic profile of adult excretory-secretory products.

The secretome of a parasite in its definitive host can be considered to be its genome in trans, to the extent that secreted products encoded by the parasite fulfill their function in the host milieu. The 'extended phenotype' of the filarial parasite, Brugia malayi, is of particular interest because of the evidence that infection results in potent down-modulation of the host immune response. We collected B. malayi 'excretory-secretory' (BES) proteins from adult parasites and using a combination of shotgun LC-MS/MS and 2D gel electrophoresis, identified 80 B. malayi and two host proteins in BES, of which 31 (38%) were detectable in whole worm extract (BmA). Products which were enriched in BES relative to BmA included phosphatidylethanolamine-binding protein (PEB), leucyl aminopeptidase (LAP, homologue of ES-62 from the related filaria Acanthocheilonema viteae), N-acetylglucosaminyltransferase (GlcNAcT) and galectin-1, in addition to the previously described major surface glycoprotein, glutathione peroxidase (gp29, GPX-1) and the cytokine homologue macrophage migration inhibitory factor (MIF-1). One of the most abundant released proteins was triose phosphate isomerase (TPI), yet many other glycolytic enzymes (such as aldolase and GAPDH) were found only in the somatic extract. Among the more prominent novel products identified in BES were a set of 11 small transthyretin-like proteins, and three glutamine-rich-repeat mucin-like proteins. Notably, no evidence was found of any secreted protein corresponding to the genome of the Wolbachia endosymbiont present in B. malayi. Western blotting with anti-phosphorylcholine (PC) monoclonal antibody identified that GlcNAcT, and not the ES-62 homologue, is the major PC-bearing protein in BES, while probing with human filariasis sera showed preferential reactivity to galectin-1 and to processed forms of myotactin. Overall, this analysis demonstrates selective release of a suite of newly identified proteins not previously suspected to be involved at the host-parasite interface, and provides important new perspectives on the biology of the filarial parasite.

TGF-ß mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus.

We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-ß. The Heligmosomoides polygyrus TGF-ß Mimic (Hp-TGM) shares no homology to any TGF-ß family member, however it binds the mammalian TGF-ß receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-ß activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation.