Immune System Investigation Using Parasitic Helminths.

Coevolutionary adaptation between humans and helminths has developed a finely tuned balance between host immunity and chronic parasitism due to immunoregulation. Given that these reciprocal forces drive selection, experimental models of helminth infection are ideally suited for discovering how host protective immune responses adapt to the unique tissue niches inhabited by these large metazoan parasites. This review highlights the key discoveries in the immunology of helminth infection made over the last decade, from innate lymphoid cells to the emerging importance of neuroimmune connections. A particular emphasis is placed on the emerging areas within helminth immunology where the most growth is possible, including the advent of genetic manipulation of parasites to study immunology and the use of engineered T cells for therapeutic options. Lastly,we cover the status of human challenge trials with helminths as treatment for autoimmune disease, which taken together, stand to keep the study of parasitic worms at the forefront of immunology for years to come.

Hookworms dynamically respond to loss of Type 2 immune pressure.

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes.

Schistosoma mansoni infection induces plasmablast and plasma cell death in the bone marrow and accelerates the decline of host vaccine responses.

Schistosomiasis is a potentially lethal parasitic disease that profoundly impacts systemic immune function in chronically infected hosts through mechanisms that remain unknown. Given the immunoregulatory dysregulation experienced in infected individuals, this study examined the impact of chronic schistosomiasis on the sustainability of vaccine-induced immunity in both children living in endemic areas and experimental infections in mice. Data show that chronic Schistosoma mansoni infection impaired the persistence of vaccine specific antibody responses in poliovirus-vaccinated humans and mice. Mechanistically, schistosomiasis primarily fostered plasmablast and plasma cell death in the bone marrow and removal of parasites following praziquantel treatment reversed the observed cell death and partially restored vaccine-induced memory responses associated with increased serum anti-polio antibody responses. Our findings strongly suggest a previously unrecognized mechanism to explain how chronic schistosomiasis interferes with an otherwise effective vaccine regimen and further advocates for therapeutic intervention strategies that reduce schistosomiasis burden in endemic areas prior to vaccination.

Transgenic expression of a T cell epitope in Strongyloides ratti reveals that helminth-specific CD4+ T cells constitute both Th2 and Treg populations.

Helminths are distinct from microbial pathogens in both size and complexity, and are the likely evolutionary driving force for type 2 immunity. CD4+ helper T cells can both coordinate worm clearance and prevent immunopathology, but issues of T cell antigen specificity in the context of helminth-induced Th2 and T regulatory cell (Treg) responses have not been addressed. Herein, we generated a novel transgenic line of the gastrointestinal nematode Strongyloides ratti expressing the immunodominant CD4+ T cell epitope 2W1S as a fusion protein with green fluorescent protein (GFP) and FLAG peptide in order to track and study helminth-specific CD4+ T cells. C57BL/6 mice infected with this stable transgenic line (termed Hulk) underwent a dose-dependent expansion of activated CD44hiCD11ahi 2W1S-specific CD4+ T cells, preferentially in the lung parenchyma. Transcriptional profiling of 2W1S-specific CD4+ T cells isolated from mice infected with either Hulk or the enteric bacterial pathogen Salmonella expressing 2W1S revealed that pathogen context exerted a dominant influence over CD4+ T cell phenotype. Interestingly, Hulk-elicited 2W1S-specific CD4+ T cells exhibited both Th2 and Treg phenotypes and expressed high levels of the EGFR ligand amphiregulin, which differed greatly from the phenotype of 2W1S-specific CD4+ T cells elicited by 2W1S-expressing Salmonella. While immunization with 2W1S peptide did not enhance clearance of Hulk infection, immunization did increase total amphiregulin production as well as the number of amphiregulin-expressing CD3+ cells in the lung following Hulk infection. Altogether, this new model system elucidates effector as well as immunosuppressive and wound reparative roles of helminth-specific CD4+ T cells. This report establishes a new resource for studying the nature and function of helminth-specific T cells.

Trefoil Factor Family: A Troika for Lung Repair and Regeneration.

Tissue damage in the upper and lower airways caused by mechanical abrasion, noxious chemicals, or pathogenic organisms must be followed by rapid restorative processes; otherwise, persistent immunopathology and disease may ensue. This review will discuss evidence for the important role served by trefoil factor (TFF) family members in healthy and diseased airways of humans and rodents. Collectively, these peptides serve to both maintain and restore homeostasis through their regulation of the mucous layer and their control of cell motility, cell differentiation, and immune function in the upper and lower airways. We will also discuss important differences in which trefoil member tracks with homeostasis and disease between humans and mice, which poses a challenge for research in this area. Moreover, we discuss new evidence supporting newly identified receptor binding partners in the leucine-rich repeat and immunoglobulin-like domain-containing NoGo (LINGO) family in mediating the biological effects of TFF proteins in mouse models of epithelial repair and infection. Recent advances in our knowledge regarding TFF peptides suggest that they may be reasonable therapeutic targets in the treatment of upper and lower airway diseases of diverse etiologies. Further work understanding their role in airway homeostasis, repair, and inflammation will benefit from these newly uncovered receptor-ligand interactions.

Myeloid-Derived IL-33 Limits the Severity of Dextran Sulfate Sodium-Induced Colitis.

IL-33 is an IL-1 family cytokine that signals through its cognate receptor, ST2, to regulate inflammation. Whether IL-33 serves a pathogenic or protective role during inflammatory bowel disease is controversial. Herein, two different strains of cell-specific conditionally deficient mice were used to compare the role of myeloid- versus intestinal epithelial cell-derived IL-33 during dextran sodium sulfate-induced colitis. Data show that loss of CD11c-restricted IL-33 exacerbated tissue pathology, coinciding with increased tissue Il6 levels and loss of intestinal forkhead box p3+ regulatory T cells. Surprisingly, the lack of intestinal epithelial cell-derived IL-33 had no impact on disease severity or tissue recovery. Thus, we show that myeloid-derived IL-33 functionally restrains colitic disease, whereas intestinal epithelial cell-derived IL-33 is dispensable.

Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection.

Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.

Oncolytic Myxoma virus infects and damages the tegument of the human parasitic flatworm Schistosoma mansoni.

Schistosomiasis is a devastating disease caused by parasitic flatworms of the genus Schistosoma. Praziquantel (PZQ), the current treatment of choice, is ineffective against immature worms and cannot prevent reinfection. The continued reliance on a single drug for treatment increases the risk of the development of PZQ-resistant parasites. Reports of PZQ insusceptibility lends urgency to the need for new therapeutics. Here, we report that Myxoma virus (MYXV), an oncolytic pox virus which is non-pathogenic in all mammals except leporids, infects and replicates in S. mansoni schistosomula, juveniles, and adult male and female worms. MYXV infection results in the shredding of the tegument and reduced egg production in vitro, identifying MYXV as the first viral pathogen of schistosomes. MYXV is currently in preclinical studies to manage multiple human cancers, supporting its use in human therapeutics. Our findings raise the exciting possibility that MYXV virus represents a novel and safe class of potential anthelmintic therapeutics.

Non-hematopoietic IL-4Rα expression contributes to fructose-driven obesity and metabolic sequelae.

OBJECTIVE: The risks of excess sugar intake in addition to high-fat diet consumption on immunopathogenesis of obesity-associated metabolic diseases are poorly defined. Interleukin-4 (IL-4) and IL-13 signaling via IL-4Rα regulates adipose tissue lipolysis, insulin sensitivity, and liver fibrosis in obesity. However, the contribution of IL-4Rα to sugar rich diet-driven obesity and metabolic sequelae remains unknown. METHODS: WT, IL-4Rα-deficient (IL-4Rα-/-) and STAT6-deficient mice (STAT6-/-) male mice were fed low-fat chow, high fat (HF) or HF plus high carbohydrate (HC/fructose) diet (HF + HC). Analysis included quantification of: (i) body weight, adiposity, energy expenditure, fructose metabolism, fatty acid oxidation/synthesis, glucose dysmetabolism and hepatocellular damage; (ii) the contribution of the hematopoietic or non-hematopoietic IL-4Rα expression; and (iii) the relevance of IL-4Rα downstream canonical STAT6 signaling pathway in this setting. RESULTS: We show that IL-4Rα regulated HF + HC diet-driven weight gain, whole body adiposity, adipose tissue inflammatory gene expression, energy expenditure, locomotor activity, glucose metabolism, hepatic steatosis, hepatic inflammatory gene expression and hepatocellular damage. These effects were potentially, and in part, dependent on non-hematopoietic IL-4Rα expression but were independent of direct STAT6 activation. Mechanistically, hepatic ketohexokinase-A and C expression was dependent on IL-4Rα, as it was reduced in IL-4Rα-deficient mice. KHK activity was also affected by HF + HC dietary challenge. Further, reduced expression/activity of KHK in IL-4Rα mice had a significant effect on fatty acid oxidation and fatty acid synthesis pathways. CONCLUSION: Our findings highlight potential contribution of non-hematopoietic IL-4Rα activation of a non-canonical signaling pathway that regulates the HF + HC diet-driven induction of obesity and severity of obesity-associated sequelae.

Tuft cells in the pathogenesis of chronic rhinosinusitis with nasal polyps and asthma.

OBJECTIVE: To review the latest discoveries regarding the role of tuft cells in the pathogenesis of chronic rhinosinusitis (CRS) with nasal polyposis and asthma. DATA SOURCES: Reviews and primary research manuscripts were identified from PubMed, Google, and bioRxiv using the search words airway epithelium, nasal polyposis, CRS or asthma and chemoreceptor cell, solitary chemosensory cell, brush cell, microvillus cell, and tuft cell. STUDY SELECTIONS: Studies were selected on the basis of novelty and likely relevance to the functions of tuft cells in chronic inflammatory diseases in the upper and lower airways. RESULTS: Tuft cells coordinate a variety of immune responses throughout the body. After the activation of bitter-taste receptors, tuft cells coordinate the secretion of antimicrobial products by adjacent epithelial cells and initiate the calcium-dependent release of acetylcholine resulting in neurogenic inflammation, including mast cell degranulation and plasma extravasation. Tuft cells are also the dominant source of interleukin-25 and a significant source of cysteinyl leukotrienes that play a role in initiating inflammatory processes in the airway. Tuft cells have also been found to seem de novo in the distal airway after a viral infection, implicating these cells in dysplastic remodeling in the distal lung in the pathogenesis of asthma. CONCLUSION: Tuft cells bridge innate and adaptive immunes responses and play an upstream role in initiating type 2 inflammation in the upper and possibly the lower airway. The role of tuft cells in respiratory pathophysiology must be further investigated, because tuft cells are putative high-value therapeutic targets for novel therapeutics in CRS with nasal polyps and asthma.

LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis.

Aim: Recovery of damaged mucosal surfaces following inflammatory insult requires diverse regenerative mechanisms that remain poorly defined. Previously, we demonstrated that the reparative actions of Trefoil Factor 3 (TFF3) depend upon the enigmatic receptor, leucine rich repeat and immunoglobulin-like domain containing nogo receptor 2 (LINGO2). This study examined the related orphan receptor LINGO3 in the context of intestinal tissue damage to determine whether LINGO family members are generally important for mucosal wound healing and maintenance of the intestinal stem cell (ISC) compartment needed for turnover of mucosal epithelium.Methods and Results: We find that LINGO3 is broadly expressed on human enterocytes and sparsely on discrete cells within the crypt niche, that contains ISCs. Loss of function studies indicate that LINGO3 is involved in recovery of normal intestinal architecture following dextran sodium sulfate (DSS)-induced colitis, and that LINGO3 is needed for therapeutic action of the long acting TFF2 fusion protein (TFF2-Fc), including a number of signaling pathways critical for cell proliferation and wound repair. LINGO3-TFF2 protein-protein interactions were relatively weak however and LINGO3 was only partially responsible for TFF2 induced MAPK signaling suggesting additional un-identified components of a receptor complex. However, deficiency in either TFF2 or LINGO3 abrogated budding/growth of intestinal organoids and reduced expression of the intestinal ISC gene leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), indicating homologous roles for these proteins in tissue regeneration, possibly via regulation of ISCs in the crypt niche.Conclusion: We propose that LINGO3 serves a previously unappreciated role in promoting mucosal wound healing.

Cell-Intrinsic Wnt4 Influences Conventional Dendritic Cell Fate Determination to Suppress Type 2 Immunity.

Whether conventional dendritic cells (cDC) acquire subset identity under direction of Wnt family glycoproteins is unknown. We demonstrate that Wnt4, a ß-catenin-independent Wnt ligand, is produced by both hematopoietic and nonhematopoietic cells and is both necessary and sufficient for preconventional DC1/cDC1 maintenance. Whereas bone marrow cDC precursors undergo phosphoJNK/c-Jun activation upon Wnt4 treatment, loss of cDC Wnt4 in CD11cCreWnt4flox/flox mice impaired differentiation of CD24+, Clec9A+, CD103+ cDC1 compared with CD11cCre controls. Conversely, single-cell RNA sequencing analysis of bone marrow revealed a 2-fold increase in cDC2 gene signature genes, and flow cytometry demonstrated increased numbers of SIRP-α+ cDC2 amid lack of Wnt4. Increased cDC2 numbers due to CD11c-restricted Wnt4 deficiency increased IL-5 production, group 2 innate lymphoid cell expansion, and host resistance to the hookworm parasite Nippostrongylus brasiliensis Collectively, these data uncover a novel and unexpected role for Wnt4 in cDC subset differentiation and type 2 immunity.

Development of solitary chemosensory cells in the distal lung after severe influenza injury.

H1N1 influenza virus infection induces dramatic and permanent alveolar remodeling mediated by p63+ progenitor cell expansion in both mice and some patients with acute respiratory distress syndrome. This persistent lung epithelial dysplasia is accompanied by chronic inflammation, but the driver(s) of this pathology are unknown. This work identified de novo appearance of solitary chemosensory cells (SCCs), as defined by the tuft cell marker doublecortin-like kinase 1, in post-influenza lungs, arising in close proximity with the dysplastic epithelium, whereas uninjured lungs are devoid of SCCs. Interestingly, fate mapping demonstrated that these cells are derived from p63-expressing lineage-negative progenitors, the same cell of origin as the dysplastic epithelium. Direct activation of SCCs with denatonium + succinate increased plasma extravasation specifically in post-influenza virus-injured lungs. Thus we demonstrate the previously unrecognized development and activity of SCCs in the lung following influenza virus infection, implicating SCCs as a central feature of dysplastic remodeling.

Trefoil Factor 2 Promotes Type 2 Immunity and Lung Repair through Intrinsic Roles in Hematopoietic and Nonhematopoietic Cells.

Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and nonhematopoietic-derived cells is essential for host protection, proliferation of alveolar type 2 cells, and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild-type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells under steady-state and infectious injury conditions.

Solitary chemosensory cells are a primary epithelial source of IL-25 in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: IL-25 can function as an early signal for the respiratory type 2 response characteristic of allergic asthma and chronic rhinosinusitis with nasal polyps (CRSwNP). In the mouse gut, tuft cells are the epithelial source of IL-25. However, the source of human airway epithelial IL-25 has remained elusive. OBJECTIVE: In this study we sought to determine whether the solitary chemosensory cell (SCC) is the predominant source of IL-25 in the sinonasal epithelium. METHOD: Flow cytometry and immunofluorescence for SCCs and IL-25 were used to interrogate polyp and turbinate tissue from patients with CRSwNP. Mucus was collected during acute inflammatory exacerbations from patients with CRSwNP or chronic rhinosinusitis without nasal polyps and IL-25 levels determined by using ELISA. Lastly, sinonasal epithelial cultures derived from polyp and turbinate tissue were stimulated with IL-13 and analyzed for SCC proliferation and IL-25 production. RESULTS: This study demonstrates that a discrete cell type, likely an SCC, characterized by expression of the taste-associated G protein gustducin and the intestinal tuft cell marker doublecortin-like kinase 1, is the predominant source of IL-25 in the human upper airway. Additionally, we show that patients with CRSwNP have increased numbers of SCCs in nasal polyp tissue and that in vitro IL-13 exposure both increased proliferation and induced apical secretion of IL-25 into the mucosal layer. CONCLUSIONS: Inflammatory sinus polyps but not adjacent turbinate tissue show expansion of the SCC population, which is the source of epithelial IL-25.

Group 2 Innate Lymphoid Cells (ILC2): Type 2 Immunity and Helminth Immunity.

Group 2 innate lymphoid cells (ILC2) have emerged as a major component of type 2 inflammation in mice and humans. ILC2 secrete large amounts of interleukins 5 and 13, which are largely responsible for host protective immunity against helminth parasites because these cytokines induce profound changes in host physiology that include: goblet cell metaplasia, mucus accumulation, smooth muscle hypercontractility, eosinophil and mast cell recruitment, and alternative macrophage activation (M2). This review covers the initial recognition of ILC2 as a distinct cell lineage, the key studies that established their biological importance, particularly in helminth infection, and the new directions that are likely to be the focus of emerging work that further explores this unique cell population in the context of health and disease.

Myeloid-Restricted AMPKα1 Promotes Host Immunity and Protects against IL-12/23p40-Dependent Lung Injury during Hookworm Infection.

How the metabolic demand of parasitism affects immune-mediated resistance is poorly understood. Immunity against parasitic helminths requires M2 cells and IL-13, secreted by CD4(+) Th2 and group 2 innate lymphoid cells (ILC2), but whether certain metabolic enzymes control disease outcome has not been addressed. This study demonstrates that AMP-activated protein kinase (AMPK), a key driver of cellular energy, regulates type 2 immunity and restricts lung injury following hookworm infection. Mice with a selective deficiency in the AMPK catalytic α1 subunit in alveolar macrophages and conventional dendritic cells produced less IL-13 and CCL17 and had impaired expansion of ILC2 in damaged lung tissue compared with wild-type controls. Defective type 2 responses were marked by increased intestinal worm burdens, exacerbated lung injury, and increased production of IL-12/23p40, which, when neutralized, restored IL-13 production and improved lung recovery. Taken together, these data indicate that defective AMPK activity in myeloid cells negatively impacts type 2 responses through increased IL-12/23p40 production. These data support an emerging concept that myeloid cells and ILC2 can coordinately regulate tissue damage at mucosal sites through mechanisms dependent on metabolic enzyme function.

JUNB is a key transcriptional modulator of macrophage activation.

Activated macrophages are crucial for restriction of microbial infection but may also promote inflammatory pathology in a wide range of both infectious and sterile conditions. The pathways that regulate macrophage activation are therefore of great interest. Recent studies in silico have putatively identified key transcription factors that may control macrophage activation, but experimental validation is lacking. In this study, we generated a macrophage regulatory network from publicly available microarray data, employing steps to enrich for physiologically relevant interactions. Our analysis predicted a novel relationship between the AP-1 family transcription factor Junb and the gene Il1b, encoding the pyrogen IL-1ß, which macrophages express upon activation by inflammatory stimuli. Previously, Junb has been characterized primarily as a negative regulator of the cell cycle, whereas AP-1 activity in myeloid inflammatory responses has largely been attributed to c-Jun. We confirmed experimentally that Junb is required for full expression of Il1b, and of additional genes involved in classical inflammation, in macrophages treated with LPS and other immunostimulatory molecules. Furthermore, Junb modulates expression of canonical markers of alternative activation in macrophages treated with IL-4. Our results demonstrate that JUNB is a significant modulator of both classical and alternative macrophage activation. Further, this finding provides experimental validation for our network modeling approach, which will facilitate the future use of gene expression data from open databases to reveal novel, physiologically relevant regulatory relationships.

Immune polarization by hookworms: taking cues from T helper type 2, type 2 innate lymphoid cells and alternatively activated macrophages.

Cellular and molecular investigation of parasitic helminth infections has greatly accelerated the understanding of type 2 immune responses. However, there remains considerable debate regarding the specific leucocytes that kill parasites and whether these mechanisms are distinct from those responsible for tissue repair. Herein, we chronicle discoveries over the past decade highlighting current paradigms in type 2 immunity with a particular emphasis upon how CD4(+) T helper type 2 cells, type 2 innate lymphoid cells and alternatively activated macrophages coordinately control helminth-induced parasitism. Primarily, this review will draw from studies of the murine nematode parasite Nippostrongylus brasiliensis, which bears important similarities to the human hookworms Ancylostoma duodenale and Necator americanus. Given that one or more hookworm species currently infect millions of individuals across the globe, we propose that vaccine and/or pharmaceutical-based cure strategies targeting these affected human populations should incorporate the conceptual advances outlined herein.