Systemic Immune Modulation by Gastrointestinal Nematodes.

Gastrointestinal nematode (GIN) infection has applied significant evolutionary pressure to the mammalian immune system and remains a global economic and human health burden. Upon infection, type 2 immune sentinels activate a common antihelminth response that mobilizes and remodels the intestinal tissue for effector function; however, there is growing appreciation of the impact GIN infection also has on the distal tissue immune state. Indeed, this effect is observed even in tissues through which GINs never transit. This review highlights how GIN infection modulates systemic immunity through (a) induction of host resistance and tolerance responses, (b) secretion of immunomodulatory products, and (c) interaction with the intestinal microbiome. It also discusses the direct consequences that changes to distal tissue immunity can have for concurrent and subsequent infection, chronic noncommunicable diseases, and vaccination efficacy.

Genetic and environmental interactions contribute to immune variation in rewilded mice.

The relative and synergistic contributions of genetics and environment to interindividual immune response variation remain unclear, despite implications in evolutionary biology and medicine. Here we quantify interactive effects of genotype and environment on immune traits by investigating C57BL/6, 129S1 and PWK/PhJ inbred mice, rewilded in an outdoor enclosure and infected with the parasite Trichuris muris. Whereas cellular composition was shaped by interactions between genotype and environment, cytokine response heterogeneity including IFNγ concentrations was primarily driven by genotype with consequence on worm burden. In addition, we show that other traits, such as expression of CD44, were explained mostly by genetics on T cells, whereas expression of CD44 on B cells was explained more by environment across all strains. Notably, genetic differences under laboratory conditions were decreased following rewilding. These results indicate that nonheritable influences interact with genetic factors to shape immune variation and parasite burden.

A new superhero, the intraepithelial mast cell, joins the "Guardians of the Gut".

The intestinal epithelium interacts with immune cells to support tissue homeostasis and coordinate responses against pathogens. In this issue of Immunity, Yang et al. unveil a central role for mast cell-epithelial cell interactions in orchestrating protective type 2 immune responses following intestinal helminth infection.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

Wriggly woes: Helminths stirring up T cell trouble.

Understanding determinants of immune response variation is central to developing treatment options. Even et al. show that naive CD4+ T cell transcriptional heterogeneity is altered by helminth infection leading to impaired immune responses independent of commensals.

Tuft cell-derived acetylcholine promotes epithelial chloride secretion and intestinal helminth clearance.

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

Tuft cell acetylcholine is released into the gut lumen to promote anti-helminth immunity.

Upon parasitic helminth infection, activated intestinal tuft cells secrete interleukin-25 (IL-25), which initiates a type 2 immune response during which lamina propria type 2 innate lymphoid cells (ILC2s) produce IL-13. This causes epithelial remodeling, including tuft cell hyperplasia, the function of which is unknown. We identified a cholinergic effector function of tuft cells, which are the only epithelial cells that expressed choline acetyltransferase (ChAT). During parasite infection, mice with epithelial-specific deletion of ChAT had increased worm burden, fitness, and fecal egg counts, even though type 2 immune responses were comparable. Mechanistically, IL-13-amplified tuft cells release acetylcholine (ACh) into the gut lumen. Finally, we demonstrated a direct effect of ACh on worms, which reduced their fecundity via helminth-expressed muscarinic ACh receptors. Thus, tuft cells are sentinels in naive mice, and their amplification upon helminth infection provides an additional type 2 immune response effector function.

The amalgam of naive CD4+ T cell transcriptional states is reconfigured by helminth infection to dampen the amplitude of the immune response.

Naive CD4+ T cells in specific pathogen-free (SPF) mice are characterized by transcriptional heterogeneity and subpopulations distinguished by the expression of quiescence, the extracellular matrix (ECM) and cytoskeleton, type I interferon (IFN-I) response, memory-like, and T cell receptor (TCR) activation genes. We demonstrate that this constitutive heterogeneity, including the presence of the IFN-I response cluster, is commensal independent insofar as being identical in germ-free and SPF mice. By contrast, Nippostrongylus brasiliensis infection altered this constitutive heterogeneity. Naive T cell-intrinsic transcriptional changes acquired during helminth infection correlated with and accounted for decreased immunization response to an unrelated antigen. These compositional and functional changes were dependent variables of helminth infection, as they disappeared at the established time point of its clearance in mice. Collectively, our results indicate that the naive T cell pool is subject to dynamic transcriptional changes in response to certain environmental cues, which in turn permutes the magnitude of the immune response.

Autoantibodies inhibit Plasmodium falciparum growth and are associated with protection from clinical malaria.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Deeper and stronger North Atlantic Gyre during the Last Glacial Maximum.

Subtropical gyre (STG) depth and strength are controlled by wind stress curl and surface buoyancy forcing1,2. Modern hydrographic data reveal that the STG extends to a depth of about 1 km in the Northwest Atlantic, with its maximum depth defined by the base of the subtropical thermocline. Despite the likelihood of greater wind stress curl and surface buoyancy loss during the Last Glacial Maximum (LGM)3, previous work suggests minimal change in the depth of the glacial STG4. Here we show a sharp glacial water mass boundary between 33° N and 36° N extending down to between 2.0 and 2.5 km-approximately 1 km deeper than today. Our findings arise from benthic foraminiferal δ18O profiles from sediment cores in two depth transects at Cape Hatteras (36-39° N) and Blake Outer Ridge (29-34° N) in the Northwest Atlantic. This result suggests that the STG, including the Gulf Stream, was deeper and stronger during the LGM than at present, which we attribute to increased glacial wind stress curl, as supported by climate model simulations, as well as greater glacial production of denser subtropical mode waters (STMWs). Our data suggest (1) that subtropical waters probably contributed to the geochemical signature of what is conventionally identified as Glacial North Atlantic Intermediate Water (GNAIW)5-7 and (2) the STG helped sustain continued buoyancy loss, water mass conversion and northwards meridional heat transport (MHT) in the glacial North Atlantic.

Ancient Plasmodium genomes shed light on the history of human malaria.

Malaria-causing protozoa of the genus Plasmodium have exerted one of the strongest selective pressures on the human genome, and resistance alleles provide biomolecular footprints that outline the historical reach of these species1. Nevertheless, debate persists over when and how malaria parasites emerged as human pathogens and spread around the globe1,2. To address these questions, we generated high-coverage ancient mitochondrial and nuclear genome-wide data from P. falciparum, P. vivax and P. malariae from 16 countries spanning around 5,500 years of human history. We identified P. vivax and P. falciparum across geographically disparate regions of Eurasia from as early as the fourth and first millennia BCE, respectively; for P. vivax, this evidence pre-dates textual references by several millennia3. Genomic analysis supports distinct disease histories for P. falciparum and P. vivax in the Americas: similarities between now-eliminated European and peri-contact South American strains indicate that European colonizers were the source of American P. vivax, whereas the trans-Atlantic slave trade probably introduced P. falciparum into the Americas. Our data underscore the role of cross-cultural contacts in the dissemination of malaria, laying the biomolecular foundation for future palaeo-epidemiological research into the impact of Plasmodium parasites on human history. Finally, our unexpected discovery of P. falciparum in the high-altitude Himalayas provides a rare case study in which individual mobility can be inferred from infection status, adding to our knowledge of cross-cultural connectivity in the region nearly three millennia ago.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is a leading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Association between telomere length and Plasmodium falciparum malaria endemicity in sub-Saharan Africans.

Leukocyte telomere length (LTL) varies significantly across human populations, with individuals of African ancestry having longer LTL than non-Africans. However, the genetic and environmental drivers of LTL variation in Africans remain largely unknown. We report here on the relationship between LTL, genetics, and a variety of environmental and climatic factors in ethnically diverse African adults (n = 1,818) originating from Botswana, Tanzania, Ethiopia, and Cameroon. We observe significant variation in LTL among populations, finding that the San hunter-gatherers from Botswana have the longest leukocyte telomeres and that the Fulani pastoralists from Cameroon have the shortest telomeres. Genetic factors explain ∼50% of LTL variation among individuals. Moreover, we observe a significant negative association between Plasmodium falciparum malaria endemicity and LTL while adjusting for age, sex, and genetics. Within Africa, adults from populations indigenous to areas with high malaria exposure have shorter LTL than those in populations indigenous to areas with low malaria exposure. Finally, we explore to what degree the genetic architecture underlying LTL in Africa covaries with malaria exposure.

Comparative genomics of Cryptosporidium parvum reveals the emergence of an outbreak-associated population in Europe and its spread to the United States.

The zoonotic parasite Cryptosporidium parvum is a global cause of gastrointestinal disease in humans and ruminants. Sequence analysis of the highly polymorphic gp60 gene enabled the classification of C. parvum isolates into multiple groups (e.g., IIa, IIc, Id) and a large number of subtypes. In Europe, subtype IIaA15G2R1 is largely predominant and has been associated with many water- and food-borne outbreaks. In this study, we generated new whole-genome sequence (WGS) data from 123 human- and ruminant-derived isolates collected in 13 European countries and included other available WGS data from Europe, Egypt, China, and the United States (n = 72) in the largest comparative genomics study to date. We applied rigorous filters to exclude mixed infections and analyzed a data set from 141 isolates from the zoonotic groups IIa (n = 119) and IId (n = 22). Based on 28,047 high-quality, biallelic genomic SNPs, we identified three distinct and strongly supported populations: Isolates from China (IId) and Egypt (IIa and IId) formed population 1; a minority of European isolates (IIa and IId) formed population 2; and the majority of European (IIa, including all IIaA15G2R1 isolates) and all isolates from the United States (IIa) clustered in population 3. Based on analyses of the population structure, population genetics, and recombination, we show that population 3 has recently emerged and expanded throughout Europe to then, possibly from the United Kingdom, reach the United States, where it also expanded. The reason(s) for the successful spread of population 3 remain elusive, although genes under selective pressure uniquely in this population were identified.

Glucose and trehalose metabolism through the cyclic pentose phosphate pathway shapes pathogen resistance and host protection in Drosophila.

Activation of immune cells requires the remodeling of cell metabolism in order to support immune function. We study these metabolic changes through the infection of Drosophila larvae by parasitoid wasp. The parasitoid egg is neutralized by differentiating lamellocytes, which encapsulate the egg. A melanization cascade is initiated, producing toxic molecules to destroy the egg while the capsule also protects the host from the toxic reaction. We combined transcriptomics and metabolomics, including 13C-labeled glucose and trehalose tracing, as well as genetic manipulation of sugar metabolism to study changes in metabolism, specifically in Drosophila hemocytes. We found that hemocytes increase the expression of several carbohydrate transporters and accordingly uptake more sugar during infection. These carbohydrates are metabolized by increased glycolysis, associated with lactate production, and cyclic pentose phosphate pathway (PPP), in which glucose-6-phosphate is re-oxidized to maximize NADPH yield. Oxidative PPP is required for lamellocyte differentiation and resistance, as is systemic trehalose metabolism. In addition, fully differentiated lamellocytes use a cytoplasmic form of trehalase to cleave trehalose to glucose and fuel cyclic PPP. Intracellular trehalose metabolism is not required for lamellocyte differentiation, but its down-regulation elevates levels of reactive oxygen species, associated with increased resistance and reduced fitness. Our results suggest that sugar metabolism, and specifically cyclic PPP, within immune cells is important not only to fight infection but also to protect the host from its own immune response and for ensuring fitness of the survivor.

The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice.

As Toxoplasma gondii disseminates through its host, the parasite must sense and adapt to its environment and scavenge nutrients. Oxygen (O2) is one such environmental factor and cytoplasmic prolyl 4-hydroxylases (PHDs) are evolutionarily conserved O2 cellular sensing proteins that regulate responses to changes in O2 availability. Toxoplasma expresses 2 PHDs. One of them, TgPHYa hydroxylates SKP1, a subunit of the SCF-E3 ubiquitin ligase complex. In vitro, TgPHYa is important for growth at low O2 levels. However, studies have yet to examine the role that TgPHYa or any other pathogen-encoded PHD plays in virulence and disease. Using a type II ME49 Toxoplasma TgPHYa knockout, we report that TgPHYa is important for Toxoplasma virulence and brain cyst formation in mice. We further find that while TgPHYa mutant parasites can establish an infection in the gut, they are unable to efficiently disseminate to peripheral tissues because the mutant parasites are unable to survive within recruited immune cells. Since this phenotype was abrogated in IFNγ knockout mice, we studied how TgPHYa mediates survival in IFNγ-treated cells. We find that TgPHYa is not required for release of parasite-encoded effectors into host cells that neutralize anti-parasitic processes induced by IFNγ. In contrast, we find that TgPHYa is required for the parasite to scavenge tryptophan, which is an amino acid whose levels are decreased after IFNγ up-regulates the tryptophan-catabolizing enzyme, indoleamine dioxygenase (IDO). We further find, relative to wild-type mice, that IDO knockout mice display increased morbidity when infected with TgPHYa knockout parasites. Together, these data identify the first parasite mechanism for evading IFNγ-induced nutritional immunity and highlight a novel role that oxygen-sensing proteins play in pathogen growth and virulence.

Role for gene conversion in the evolution of cell-surface antigens of the malaria parasite Plasmodium falciparum.

While the malaria parasite Plasmodium falciparum has low average genome-wide diversity levels, likely due to its recent introduction from a gorilla-infecting ancestor (approximately 10,000 to 50,000 years ago), some genes display extremely high diversity levels. In particular, certain proteins expressed on the surface of human red blood cell-infecting merozoites (merozoite surface proteins (MSPs)) possess exactly 2 deeply diverged lineages that have seemingly not recombined. While of considerable interest, the evolutionary origin of this phenomenon remains unknown. In this study, we analysed the genetic diversity of 2 of the most variable MSPs, DBLMSP and DBLMSP2, which are paralogs (descended from an ancestral duplication). Despite thousands of available Illumina WGS datasets from malaria-endemic countries, diversity in these genes has been hard to characterise as reads containing highly diverged alleles completely fail to align to the reference genome. To solve this, we developed a pipeline leveraging genome graphs, enabling us to genotype them at high accuracy and completeness. Using our newly- resolved sequences, we found that both genes exhibit 2 deeply diverged lineages in a specific protein domain (DBL) and that one of the 2 lineages is shared across the genes. We identified clear evidence of nonallelic gene conversion between the 2 genes as the likely mechanism behind sharing, leading us to propose that gene conversion between diverged paralogs, and not recombination suppression, can generate this surprising genealogy; a model that is furthermore consistent with high diversity levels in these 2 genes despite the strong historical P. falciparum transmission bottleneck.

Role of Rabenosyn-5 and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites.

Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of Plasmodium falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite's food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the repurposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here, we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5L and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.