Intestinal cell diversity and treatment responses in a parasitic nematode at single cell resolution.

BACKGROUND: Parasitic nematodes, significant pathogens for humans, animals, and plants, depend on diverse organ systems for intra-host survival. Understanding the cellular diversity and molecular variations underlying these functions holds promise for developing novel therapeutics, with specific emphasis on the neuromuscular system's functional diversity. The nematode intestine, crucial for anthelmintic therapies, exhibits diverse cellular phenotypes, and unraveling this diversity at the single-cell level is essential for advancing knowledge in anthelmintic research across various organ systems. RESULTS: Here, using novel single-cell transcriptomics datasets, we delineate cellular diversity within the intestine of adult female Ascaris suum, a parasitic nematode species that infects animals and people. Gene transcripts expressed in individual nuclei of untreated intestinal cells resolved three phenotypic clusters, while lower stringency resolved additional subclusters and more potential diversity. Clusters 1 and 3 phenotypes displayed variable congruence with scRNA phenotypes of C. elegans intestinal cells, whereas the A. suum cluster 2 phenotype was markedly unique. Distinct functional pathway enrichment characterized each A. suum intestinal cell cluster. Cluster 2 was distinctly enriched for Clade III-associated genes, suggesting it evolved within clade III nematodes. Clusters also demonstrated differential transcriptional responsiveness to nematode intestinal toxic treatments, with Cluster 2 displaying the least responses to short-term intra-pseudocoelomic nematode intestinal toxin treatments. CONCLUSIONS: This investigation presents advances in knowledge related to biological differences among major cell populations of adult A. suum intestinal cells. For the first time, diverse nematode intestinal cell populations were characterized, and associated biological markers of these cells were identified to support tracking of constituent cells under experimental conditions. These advances will promote better understanding of this and other parasitic nematodes of global importance, and will help to guide future anthelmintic treatments.

NF-κB signaling deficiency in CD11c-expressing phagocytes mediates early inflammatory responses and enhances Mycobacterium tuberculosis control.

Early innate immune responses play an important role in determining the protective outcome of Mycobacterium tuberculosis (Mtb) infection. Nuclear factor kappa B (NF-κB) signaling in immune cells regulates the expression of key downstream effector molecules that mount early anti-mycobacterial responses. Using conditional knockout mice, we studied the effect of abrogation of NF-κB signaling in different myeloid cell types and its impact on Mtb infection. Our results show that absence of IKK2-mediated signaling in all myeloid cells resulted in increased susceptibility to Mtb infection. In contrast, absence of IKK2-mediated signaling specifically in CD11c+ myeloid cells induced early pro-inflammatory cytokine responses, enhanced the recruitment of myeloid cells and mediated early resistance to Mtb. Abrogation of IKK2 in MRP8-expressing neutrophils did not impact either disease pathology or Mtb control. Thus, we describe an early immunoregulatory role for NF-κB signaling in CD11c-expressing phagocytes, and a later protective role for NF-κB in LysM-expressing cells during Mtb infection.

Social and psychological adversity are associated with distinct mother and infant gut microbiome variations.

Health disparities are driven by underlying social disadvantage and psychosocial stressors. However, how social disadvantage and psychosocial stressors lead to adverse health outcomes is unclear, particularly when exposure begins prenatally. Variations in the gut microbiome and circulating proinflammatory cytokines offer potential mechanistic pathways. Here, we interrogate the gut microbiome of mother-child dyads to compare high-versus-low prenatal social disadvantage, psychosocial stressors and maternal circulating cytokine cohorts (prospective case-control study design using gut microbiomes from 121 dyads profiled with 16 S rRNA sequencing and 89 dyads with shotgun metagenomic sequencing). Gut microbiome characteristics significantly predictive of social disadvantage and psychosocial stressors in the mothers and children indicate that different discriminatory taxa and related pathways are involved, including many species of Bifidobacterium and related pathways across several comparisons. The lowest inter-individual gut microbiome similarity was observed among high-social disadvantage/high-psychosocial stressors mothers, suggesting distinct environmental exposures driving a diverging gut microbiome assembly compared to low-social disadvantage/low-psychosocial stressors controls (P = 3.5 × 10-5 for social disadvantage, P = 2.7 × 10-15 for psychosocial stressors). Children's gut metagenome profiles at 4 months also significantly predicted high/low maternal prenatal IL-6 (P = 0.029), with many bacterial species overlapping those identified by social disadvantage and psychosocial stressors. These differences, based on maternal social and psychological status during a critical developmental window early in life, offer potentially modifiable targets to mitigate health inequities.

The proteome of extracellular vesicles of the lung fluke Paragonimus kellicotti produced in vitro and in the lung cyst.

Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by Paragonimus kellicotti adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils. The majority of EV were approximately 30-50 nm in diameter. We identified 548 P. kellicotti-derived proteins in EV ESP by mass spectrometry and 8 proteins in EV CFP of which 7 were also present in EV ESP. No parasite-derived proteins were reliably detected in EV isolated from plasma samples. A cysteine protease (MK050848, CP-6) was the most abundant protein found in EV CFP in all technical and biological replicates. Immunolocalization of CP-6 showed strong labeling in the tegument of P. kellicotti and in the adjacent cyst and lung tissue that contained worm eggs. It is likely that CP-6 present in EV is involved in parasite-host interactions. These results provide new insights into interactions between Paragonimus and their mammalian hosts, and they provide potential clues for development of novel diagnostic tools and treatments.

Poly(ADP-ribose) polymerase 9 mediates early protection against Mycobacterium tuberculosis infection by regulating type I IFN production.

The ADP ribosyltransferases (PARPs 1-17) regulate diverse cellular processes, including DNA damage repair. PARPs are classified on the basis of their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). Although PARP9 mRNA expression is significantly increased in progressive tuberculosis (TB) in humans, its participation in host immunity to TB is unknown. Here, we show that PARP9 mRNA encoding the MARylating PARP9 enzyme was upregulated during TB in humans and mice and provide evidence of a critical modulatory role for PARP9 in DNA damage, cyclic GMP-AMP synthase (cGAS) expression, and type I IFN production during TB. Thus, Parp9-deficient mice were susceptible to Mycobacterium tuberculosis infection and exhibited increased TB disease, cGAS and 2'3'-cyclic GMP-AMP (cGAMP) expression, and type I IFN production, along with upregulation of complement and coagulation pathways. Enhanced M. tuberculosis susceptibility is type I IFN dependent, as blockade of IFN α receptor (IFNAR) signaling reversed the enhanced susceptibility of Parp9-/- mice. Thus, in sharp contrast to PARP9 enhancement of type I IFN production in viral infections, this member of the MAR family plays a protective role by limiting type I IFN responses during TB.

Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm's excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

Direct Proteomic Detection and Prioritization of 19 Onchocerciasis Biomarker Candidates in Humans.

Onchocerca volvulus, the causative agent of onchocerciasis, infects over 20 million people and can cause severe dermatitis and ocular conditions including blindness. Current treatments employed in mass drug administration programs do not kill adult female worms, and common diagnostic tests cannot reliably assess viability of adult worms. There is an urgent need for better diagnostic tests to facilitate monitoring the efficacy of new treatments and disease elimination efforts. Here, eight plasma samples collected from individuals infected with O. volvulus and seven from uninfected individuals were analyzed by MS/MS spectrometry to directly identify O. volvulus proteins present in infected but absent in uninfected control samples. This direct proteomic approach for biomarker discovery had not been previously employed for onchocerciasis. Among all detected proteins, 19 biomarker candidates were supported by two or more unique peptides, identified in the plasma of at least three O. volvulus-infected human samples and absent in all control samples. Comprehensive analysis and ranking of these candidates included detailed functional annotation and a review of RNA-seq gene expression profiles. Isotope-labeled standard peptides were run in parallel and validated MS/MS peptide identifications for 15 peptides from 11 of the 19 proteins, and two infected urine and one uninfected urine sample was used for additional validation. A major antigen/OVOC11613 was identified as the most promising candidate with eight unique peptides across five plasma samples and one urine sample. Additional strong candidates included OVOC1523/ATP synthase, OVOC247/laminin and OVOC11626/PLK5, and along with OVOC11613, and were also detected in urine samples from onchocerciasis patients. This study has identified a promising novel set of proteins that will be carried forward to develop assays that can be used for diagnosis of O. volvulus infections and for monitoring treatment efficacy.

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia.

Enterotoxigenic E. coli (ETEC) produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and commonly cause diarrhea in resource-poor regions. ETEC have been linked repeatedly to sequelae in children including enteropathy, malnutrition, and growth impairment. Although cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their contributions to sequelae remain unclear. LT increases cellular cAMP to activate protein kinase A (PKA) that phosphorylates ion channels driving intestinal export of salt and water resulting in diarrhea. As PKA also modulates transcription of many genes, we interrogated transcriptional profiles of LT-treated intestinal epithelia. Here we show that LT significantly alters intestinal epithelial gene expression directing biogenesis of the brush border, the major site for nutrient absorption, suppresses transcription factors HNF4 and SMAD4 critical to enterocyte differentiation, and profoundly disrupts microvillus architecture and essential nutrient transport. In addition, ETEC-challenged neonatal mice exhibit substantial brush border derangement that is prevented by maternal vaccination with LT. Finally, mice repeatedly challenged with toxigenic ETEC exhibit impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to prevent major sequelae of these common infections including malnutrition that impact millions of children.

The gut microbial metabolic capacity of microbiome-humanized vs. wild type rodents reveals a likely dual role of intestinal bacteria in hepato-intestinal schistosomiasis.

Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation of mechanisms aimed to prevent excessive injuries caused by migrating parasite eggs. Complementing data from previous studies, our findings suggest that the host gut microbiome might play a dual role in the pathophysiology of schistosomiasis, where intestinal bacteria may contribute to egg-associated pathology while, in turn, protect the host from uncontrolled tissue damage.

Evolution of Multiple Domains of the HIV-1 Envelope Glycoprotein during Coreceptor Switch with CCR5 Antagonist Therapy.

HIV-1 uses CD4 as a receptor and chemokine receptors CCR5 and/or CXCR4 as coreceptors. CCR5 antagonists are a class of antiretrovirals used to inhibit viral entry. Phenotypic prediction algorithms such as Geno2Pheno are used to assess CCR5 antagonist eligibility, for which the V3 region is screened. However, there exist scenarios where the algorithm cannot give an accurate prediction of tropism. The current study examined coreceptor shift of HIV-1 from CCR5-tropic strains to CXCR4-tropic or dual-tropic strains among five subjects in a clinical trial of the CCR5 antagonist vicriviroc. Envelope gene amplicon libraries were constructed and subjected to next-generation sequencing, as well as single-clone sequencing and functional analyses. Approximately half of the amplified full-length single envelope-encoding clones had no significant activity for infection of cells expressing high levels of CD4 and CCR5 or CXCR4. Functional analysis of 9 to 21 individual infectious clones at baseline and at the time of VF were used to construct phylogenetic trees and sequence alignments. These studies confirmed that specific residues and the overall charge of the V3 loop were the major determinants of coreceptor use, in addition to specific residues in other domains of the envelope protein in V1/V2, V4, C3, and C4 domains that may be important for coreceptor shift. These results provide greater insight into the viral genetic determinants of coreceptor shift. IMPORTANCE This study is novel in combining single-genome sequence analysis and next-generation sequencing to characterize HIV-1 quasispecies. The work highlights the importance of mutants present at frequencies of 1% or less in development of drug resistance. This study highlights a critical role of specific amino acid substitutions outside V3 that contribute to coreceptor shift as well as important roles of the V1/V2, V4, C3, and C4 domain residues.

Aspartyl Protease Inhibitors as Anti-Filarial Drugs.

The current treatments for lymphatic filariasis and onchocerciasis do not effectively kill the adult parasitic nematodes, allowing these chronic and debilitating diseases to persist in millions of people. Thus, the discovery of new drugs with macrofilaricidal potential to treat these filarial diseases is critical. To facilitate this need, we first investigated the effects of three aspartyl protease inhibitors (APIs) that are FDA-approved as HIV antiretroviral drugs on the adult filarial nematode, Brugia malayi and the endosymbiotic bacteria, Wolbachia. From the three hits, nelfinavir had the best potency with an IC50 value of 7.78 µM, followed by ritonavir and lopinavir with IC50 values of 14.3 µM and 16.9 µM, respectively. The three APIs have a direct effect on killing adult B. malayi after 6 days of exposure in vitro and did not affect the Wolbachia titers. Sequence conservation and stage-specific gene expression analysis identified Bm8660 as the most likely primary aspartic protease target for these drug(s). Immunolocalization using antibodies raised against the Bm8660 ortholog of Onchocerca volvulus showed it is strongly expressed in female B. malayi, especially in metabolically active tissues such as lateral and dorsal/ventral chords, hypodermis, and uterus tissue. Global transcriptional response analysis using adult female B. pahangi treated with APIs identified four additional aspartic proteases differentially regulated by the three effective drugs, as well as significant enrichment of various pathways including ubiquitin mediated proteolysis, protein kinases, and MAPK/AMPK/FoxO signaling. In vitro testing against the adult gastro-intestinal nematode Trichuris muris suggested broad-spectrum potential for these APIs. This study suggests that APIs may serve as new leads to be further explored for drug discovery to treat parasitic nematode infections.

Horizontal gene transfer provides insights into the deep evolutionary history and biology of Trichinella.

Evolution involves temporal changes in the characteristics of a species that are subsequently propagated or rejected through natural selection. In the case of parasites, host switching also plays a prominent role in the evolutionary process. These changes are rooted in genetic variation and gene flow where genes may be deleted, mutated (sequence), duplicated, rearranged and/or translocated and then transmitted through vertical gene transfer. However, the introduction of new genes is not driven only by Mendelian inheritance and mutation but also by the introduction of DNA from outside a lineage in the form of horizontal gene transfer between donor and recipient organisms. Once introduced and integrated into the biology of the recipient, vertical inheritance then perpetuates the newly acquired genetic factor, where further functionality may involve co-option of what has become a pre-existing physiological capacity. Upon sequencing the Trichinella spiralis (Clade I) genome, a cyanate hydratase (cyanase) gene was identified that is common among bacteria, fungi, and plants, but rarely observed among other eukaryotes. The sequence of the Trichinella cyanase gene clusters with those derived from the Kingdom Plantae in contrast to the genes found in some Clade III and IV nematodes that cluster with cyanases of bacterial origin. Phylogenetic analyses suggest that the Trichinella cyanase was acquired during the Devonian period and independently from those of other nematodes. These data may help inform us of the deep evolutionary history and ecological connectivity of early ancestors within the lineage of contemporary Trichinella. Further, in many extant organisms, cyanate detoxification has been largely superseded by energy requirements for metabolism. Thus, deciphering the function of Trichinella cyanase may provide new avenues for treatment and control.

Phylum-Spanning Neuropeptide GPCR Identification and Prioritization: Shaping Drug Target Discovery Pipelines for Nematode Parasite Control.

Nematode parasites undermine human health and global food security. The frontline anthelmintic portfolio used to treat parasitic nematodes is threatened by the escalation of anthelmintic resistance, resulting in a demand for new drug targets for parasite control. Nematode neuropeptide signalling pathways represent an attractive source of novel drug targets which currently remain unexploited. The complexity of the nematode neuropeptidergic system challenges the discovery of new targets for parasite control, however recent advances in parasite 'omics' offers an opportunity for the in silico identification and prioritization of targets to seed anthelmintic discovery pipelines. In this study we employed Hidden Markov Model-based searches to identify ~1059 Caenorhabditis elegans neuropeptide G-protein coupled receptor (Ce-NP-GPCR) encoding gene homologs in the predicted protein datasets of 10 key parasitic nematodes that span several phylogenetic clades and lifestyles. We show that, whilst parasitic nematodes possess a reduced complement of Ce-NP-GPCRs, several receptors are broadly conserved across nematode species. To prioritize the most appealing parasitic nematode NP-GPCR anthelmintic targets, we developed a novel in silico nematode parasite drug target prioritization pipeline that incorporates pan-phylum NP-GPCR conservation, C. elegans-derived reverse genetics phenotype, and parasite life-stage specific expression datasets. Several NP-GPCRs emerge as the most attractive anthelmintic targets for broad spectrum nematode parasite control. Our analyses have also identified the most appropriate targets for species- and life stage- directed chemotherapies; in this context we have identified several NP-GPCRs with macrofilaricidal potential. These data focus functional validation efforts towards the most appealing NP-GPCR targets and, in addition, the prioritization strategy employed here provides a blueprint for parasitic nematode target selection beyond NP-GPCRs.

Ascaris suum Informs Extrasynaptic Volume Transmission in Nematodes.

Neural circuit synaptic connectivities (the connectome) provide the anatomical foundation for our understanding of nematode nervous system function. However, other nonsynaptic routes of communication are known in invertebrates including extrasynaptic volume transmission (EVT), which enables short- and/or long-range communication in the absence of synaptic connections. Although EVT has been highlighted as a facet of Caenorhabditis elegans neurosignaling, no experimental evidence identifies body cavity fluid (pseudocoelomic fluid; PCF) as a vehicle for either neuropeptide or biogenic amine transmission. In the parasitic nematode Ascaris suum, FMRFamide-like peptides encoded on flp-18 potently stimulate female reproductive organs but are expressed in cells that are anatomically distant from the reproductive organ, with no known synaptic connections to this tissue. Here we investigate nonsynaptic neuropeptide signaling in nematodes mediated by the body cavity fluid. Our data show that (i) A. suum PCF (As-PCF) contains a catalog of neuropeptides including FMRFamide-like peptides and neuropeptide-like proteins, (ii) the A. suum FMRFamide-like peptide As-FLP-18A dominates the As-PCF peptidome, (iii) As-PCF potently modulates nematode reproductive muscle function ex vivo, mirroring the effects of synthetic FLP-18 peptides, (iv) As-PCF activates the C. elegans FLP-18 receptors NPR-4 and -5, (v) As-PCF alters C. elegans behavior, and (vi) FLP-18 and FLP-18 receptors display pan-phylum distribution in nematodes. This study provides the first direct experimental evidence to support an extrasynaptic volume route for neuropeptide transmission in nematodes. These data indicate nonsynaptic signaling within the nematode functional connectome and are particularly pertinent to receptor deorphanization approaches underpinning drug discovery programs for nematode pathogens.

Cell Death and Transcriptional Responses Induced in Larvae of the Nematode Haemonchus contortus by Toxins/Toxicants with Broad Phylogenetic Efficacy.

Establishing methods to investigate treatments that induce cell death in parasitic nematodes will promote experimental approaches to elucidate mechanisms and to identify prospective anthelmintics capable of inducing this outcome. Here, we extended recent progress on a method to monitor cell death and to identify small molecule inhibitors in Ascaris suum to Haemonchus contortus, a phylogenetically distant parasitic nematode of significance for both human and agricultural animal health. We utilized a diverse group of small molecule inhibitors referred to as nematode intestinal toxins/toxicants (NITs) coupled with motility, cytological and cell death assays to resolve gross effects on motility and individual cells and organ systems of two H. contortus larval stages in culture. Early transcriptional response evaluation identified NIT-responsive genes and pathways. The scope of death among cells in larvae varied among NITs but shared patterns with A. suum, despite the approach having some limitations due to characteristics of H. contortus larvae. Gene response patterns varied among NITs tested and provided information on the cell targets and pathways affected. Experimental NIT assays provide tools capable of inducing cell death in larval stages of parasitic nematodes, and can resolve many individual cells and organ systems in which cell death can be induced.

Lung Epithelial Signaling Mediates Early Vaccine-Induced CD4+ T Cell Activation and Mycobacterium tuberculosis Control.

Tuberculosis (TB) is one of the leading causes of death due to a single infectious agent. The development of a TB vaccine that induces durable and effective immunity to Mycobacterium tuberculosis (Mtb) infection is urgently needed. Early and superior Mtb control can be induced in M. bovis Bacillus Calmette-Guérin (BCG)-vaccinated hosts when the innate immune response is targeted to generate effective vaccine-induced immunity. In the present study, we show that innate activation of DCs is critical for mucosal localization of clonally activated vaccine-induced CD4+ T cells in the lung and superior early Mtb control. In addition, our study reveals that Th1/Th17 cytokine axis play an important role in superior vaccine-induced immunity. Our studies also show that activation of the nuclear factor kappa-light-chain enhancer of activated B cell (NF-κß) pathway in lung epithelial cells is critical for the mucosal localization of activated vaccine-induced CD4+ T cells for rapid Mtb control. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control. IMPORTANCE Tuberculosis is a leading cause of death due to single infectious agent accounting 1.4 million deaths each year. The only licensed vaccine, BCG, is not effective due to variable efficacy. In our study, we determined the early immune events necessary for achieving complete protection in a BCG-vaccinated host. Our study reveals that innate activation of DCs can mediate superior and early Mtb control in BCG-vaccinated mice through lung epithelial cell signaling and localization of clonal activated, Mtb antigen-specific, cytokine-producing CD4+ T cells within the lung parenchyma and airways. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control.

Whipworm-Associated Intestinal Microbiome Members Consistent Across Both Human and Mouse Hosts.

The human whipworm Trichuris trichiura infects 289 million people worldwide, resulting in substantial morbidity. Whipworm infections are difficult to treat due to low cure rates and high reinfection rates. Interactions between whipworm and its host's intestinal microbiome present a potential novel target for infection control or prevention but are very complicated and are identified using inconsistent methodology and sample types across the literature, limiting their potential usefulness. Here, we used a combined 16S rRNA gene OTU analysis approach (QIIME2) for samples from humans and mice infected with whipworm (T. trichiura and T. muris, respectively) to identify for the first time, bacterial taxa that were consistently associated with whipworm infection spanning host species and infection status using four independent comparisons (baseline infected vs uninfected and before vs after deworming for both humans and mice). Using these four comparisons, we identified significant positive associations for seven taxa including Escherichia, which has been identified to induce whipworm egg hatching, and Bacteroides, which has previously been identified as a major component of the whipworm internal microbiome. We additionally identified significant negative associations for five taxa including four members of the order Clostridiales, two from the family Lachnospiraceae, including Blautia which was previously identified as positively associated with whipworm in independent human and mouse studies. Using this approach, bacterial taxa of interest for future association and mechanistic studies were identified, and several were validated by RT-qPCR. We demonstrate the applicability of a mouse animal model for comparison to human whipworm infections with respect to whipworm-induced intestinal microbiome disruption and subsequent restoration following deworming. Overall, the novel cross-species analysis approach utilized here provides a valuable research tool for studies of the interaction between whipworm infection and the host intestinal microbiome.

IFN signaling and neutrophil degranulation transcriptional signatures are induced during SARS-CoV-2 infection.

SARS-CoV-2 virus has infected more than 92 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Using a rhesus macaque model of SARS-CoV-2 infection, we have characterized the transcriptional signatures induced in the lungs of juvenile and old macaques following infection. Genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated, as also seen in lungs of macaques with tuberculosis. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. Together, our transcriptomic studies have delineated disease pathways that improve our understanding of the immunopathogenesis of COVID-19.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity.

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.