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Distinct subsets of T lymphocytes express CX3CR1 under inflammatory conditions, but little is known about CX3CR1+CD4+ T cells during type 2 inflammation in helminth infections. In this study, we used a fate-mapping mouse model to characterize CX3CR1+CD4+ T cells during both acute Nippostrongylus brasiliensis and chronic Schistosoma mansoni murine models of helminth infections, revealing CX3CR1+CD4+ T cells to be an activated tissue-homing subset with varying capacity for cytokine production. Tracking these cells over time revealed that maintenance of CX3CR1 itself along with a TH2 phenotype conferred a survival advantage in the inflamed tissue. Single-cell RNA sequencing analysis of fate-mapped CX3CR1+CD4+ T cells from both the peripheral tissue and the spleen revealed a considerable level of diversity and identified a distinct population of BCL6+TCF-1+PD1+CD4+ T cells in the spleen during helminth infections. Conditional deletion of BCL6 in CX3CR1+ cells resulted in fewer CX3CR1+CD4+ T cells during infection, indicating a role in sustaining CD4+ T cell responses to helminth infections. Overall, our studies revealed the behavior and heterogeneity of CX3CR1+CD4+ T cells during type 2 inflammation in helminth infections and identified BCL6 to be important in their maintenance., (Copyright © 2024 by The American Association of Immunologists, Inc.)

SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αβ T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection., Competing Interests: M.J.M. reported potential competing interests: laboratory research and clinical trials contracts with Lilly, Pfizer (exclusive of the current work), and Sanofi for vaccines or MAB vs. SARS-CoV-2; contract funding from USG/HHS/BARDA for research specimen characterization and repository; research grant funding from USG/HHS/NIH for SARS-CoV-2 vaccine and MAB clinical trials; personal fees from Meissa Vaccines, Inc. and Pfizer for Scientific Advisory Board service. RSH has received research support from CareDx for SARS-CoV-2 vaccine studies and has performed consulting work for Bristol-Myers-Squibb., (© 2023 The Authors.)

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and Abbvie; consulted for or received an honoraria from Puretech Health, Genentech, and Abbvie; and is an inventor on U.S. patent 10,722,600 and provisional patent 62/935,035 and 63/157,225, (Copyright: © 2023 Robertson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Our previous studies identified a population of stem cell-like proliferating myeloid cells within inflamed tissues that could serve as a reservoir for tissue macrophages to adopt different activation states depending on the microenvironment. By lineage-tracing cells derived from CX3CR1+ precursors in mice during infection and profiling by single-cell RNA sequencing, in this study, we identify a cluster of BIRC5+ myeloid cells that expanded in the liver during chronic infection with either the parasite Schistosoma mansoni or the bacterial pathogen Staphylococcus aureus. In the absence of tissue-damaging toxins, S. aureus infection does not elicit these BIRC5+ cells. Moreover, deletion of BIRC5 from CX3CR1-expressing cells results in improved survival during S. aureus infection. Hence the combination of single-cell RNA sequencing and genetic fate-mapping CX3CR1+ cells revealed a toxin-dependent pathogenic role for BIRC5 in myeloid cells during S. aureus infection., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Following activation by cognate antigen, B cells undergo fine-tuning of their antigen receptors and may ultimately differentiate into antibody-secreting cells (ASCs). While antigen-specific B cells that express surface receptors (B cell receptors [BCRs]) can be readily cloned and sequenced following flow sorting, antigen-specific ASCs that lack surface BCRs cannot be easily profiled. Here, we report an approach, TRAPnSeq (antigen specificity mapping through immunoglobulin [Ig] secretion TRAP and Sequencing), that allows capture of secreted antibodies on the surface of ASCs, which in turn enables high-throughput screening of single ASCs against large antigen panels. This approach incorporates flow cytometry, standard microfluidic platforms, and DNA-barcoding technologies to characterize antigen-specific ASCs through single-cell V(D)J, RNA, and antigen barcode sequencing. We show the utility of TRAPnSeq by profiling antigen-specific IgG and IgE ASCs from both mice and humans and highlight its capacity to accelerate therapeutic antibody discovery from ASCs., Competing Interests: This study was sponsored by Regeneron Pharmaceuticals, Inc. All authors are current or former employees of Regeneron and may hold stock options in the company. S.A., J.C.D., A.V., B.K., I.S., G.S.A., M.A.S., W.K.L., and J.M.O. are inventors on a pending US patent application on TRAPnSeq (“Methods of Mapping Antigen Specificity to Antibody-Secreting Cells”). A.J.M. is an inventor on a pending US patent application (#16/363,774; “Humanized Rodents for Testing Therapeutic Agents”)., (© 2023 The Authors.)

Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle., Competing Interests: Declaration of interests K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and Abbvie, consulted for or received honoraria from Vedanta, Genentech, and Abbvie, and is an inventor on US patent 10,722,600 and provisional patents 62/935,035 and 63/157,225., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cerebral malaria is a severe complication of Plasmodium falciparum infection characterized by the loss of blood-brain barrier (BBB) integrity, which is associated with brain swelling and mortality in patients. P. falciparum-infected red blood cells and inflammatory cytokines, like tumor necrosis factor alpha (TNF-α), have been implicated in the development of cerebral malaria, but it is still unclear how they contribute to the loss of BBB integrity. Here, a combination of transcriptomic analysis and cellular assays detecting changes in barrier integrity and endothelial activation were used to distinguish between the effects of P. falciparum and TNF-α on a human brain microvascular endothelial cell (HBMEC) line and in primary human brain microvascular endothelial cells. We observed that while TNF-α induced high levels of endothelial activation, it only caused a small increase in HBMEC permeability. Conversely, P. falciparum-infected red blood cells (iRBCs) led to a strong increase in HBMEC permeability that was not mediated by cell death. Distinct transcriptomic profiles of TNF-α and P. falciparum in HBMECs confirm the differential effects of these stimuli, with the parasite preferentially inducing an endoplasmic reticulum stress response. Our results establish that there are fundamental differences in the responses induced by TNF-α and P. falciparum on brain endothelial cells and suggest that parasite-induced signaling is a major component driving the disruption of the BBB during cerebral malaria, proposing a potential target for much needed therapeutics. IMPORTANCE Cerebral malaria is a severe complication of Plasmodium falciparum infection that causes the loss of blood-brain barrier integrity and frequently results in death. Here, we compared the effect of P. falciparum-infected red blood cells and inflammatory cytokines, like TNF-α, in the loss of BBB integrity. We observed that while TNF-α induced a small increase in barrier permeability, P. falciparum-infected red blood cells led to a severe loss of barrier integrity. Our results establish that there are fundamental differences in the responses induced by TNF-α and P. falciparum on brain endothelial cells and suggest that parasite-induced signaling is a major component driving the disruption of the BBB during cerebral malaria, proposing a potential target for much needed therapeutics.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease 1-7 . Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium 8,9 . This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Staphylococcus aureus is an opportunistic pathogen and chief among bloodstream-infecting bacteria. S. aureus produces an array of human-specific virulence factors that may contribute to immune suppression. Here, we defined the response of primary human phagocytes following infection with S. aureus using RNA-sequencing (RNA-Seq). We found that the overall transcriptional response to S. aureus was weak both in the number of genes and in the magnitude of response. Using an ex vivo bacteremia model with fresh human blood, we uncovered that infection with S. aureus resulted in the down-regulation of genes related to innate immune response and cytokine and chemokine signaling. This muted transcriptional response was conserved across diverse S. aureus clones but absent in blood exposed to heat-killed S. aureus or blood infected with the less virulent staphylococcal species Staphylococcus epidermidis . Notably, this signature was also present in patients with S. aureus bacteremia. We identified the master regulator S. aureus exoprotein expression (SaeRS) and the SaeRS-regulated pore-forming toxins as key mediators of the transcriptional suppression. The S. aureus -mediated suppression of chemokine and cytokine transcription was reflected by circulating protein levels in the plasma. Wild-type S. aureus elicited a soluble milieu that was restrictive in the recruitment of human neutrophils compared with strains lacking saeRS . Thus, S. aureus blunts the inflammatory response resulting in impaired neutrophil recruitment, which could promote the survival of the pathogen during invasive infection.

Trichuris nematodes reproduce within the microbiota-rich mammalian intestine and lay thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Here, we show that bacterial byproducts facilitate the reproductive development of nematodes. First, we employed a pipeline using the well-characterized, free-living nematode C. elegans to identify microbial factors with conserved roles in nematode reproduction. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Additionally, Trichuris muris eggs displayed defective hatching in the presence of fabH- or eutN-deficient E. coli due to reduced arginine or elevated aldehydes, respectively. T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed morphological defects and failed to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly related nematodes., Competing Interests: Declaration of interests K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech, and Abbvie. K.C. has consulted for or received honoraria from Puretech Health, Genentech, and Abbvie. K.C. holds U.S. patent 10,722,600 and provisional patent 62/935,035 and 63/157,225, and E.J.A.H. holds US patent 6,087,153., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury., Competing Interests: Declaration of interests Z.W. and S.L.H. report being named as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, Quest Diagnostics, and Procter & Gamble. S.L.H. also reports being a paid consultant for Procter & Gamble, having received research funds from Procter & Gamble and Roche Diagnostics, and being eligible to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland HeartLab, Quest Diagnostics, and Procter & Gamble. The other authors report that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background & Aims: Restorative proctocolectomy with ileal pouch-anal anastomosis is a surgical procedure in patients with ulcerative colitis refractory to medical therapies. Pouchitis, the most common complication, is inflammation of the pouch of unknown etiology. To define how the intestinal immune system is distinctly organized during pouchitis, we analyzed tissues from patients with and without pouchitis and from patients with ulcerative colitis using single-cell RNA sequencing (scRNA-seq)., Methods: We examined pouch lamina propria CD45+ hematopoietic cells from intestinal tissues of ulcerative colitis patients with (n = 15) and without an ileal pouch-anal anastomosis (n = 11). Further in silico meta-analysis was performed to generate transcriptional interaction networks and identify biomarkers for patients with inflamed pouches., Results: In addition to tissue-specific signatures, we identified a population of IL1B/LYZ+ myeloid cells and FOXP3/BATF+ T cells that distinguish inflamed tissues, which we further validated in other scRNA-seq datasets from patients with inflammatory bowel disease (IBD). Cell-type-specific transcriptional markers obtained from scRNA-seq was used to infer representation from bulk RNA sequencing datasets, which further implicated myeloid cells expressing IL1B and S100A8/A9 calprotectin as interacting with stromal cells, and Bacteroidales and Clostridiales bacterial taxa. We found that nonresponsiveness to anti-integrin biologic therapies in patients with ulcerative colitis was associated with the signature of IL1B+/LYZ+ myeloid cells in a subset of patients., Conclusions: Features of intestinal inflammation during pouchitis and ulcerative colitis are similar, which may have clinical implications for the management of pouchitis. scRNA-seq enables meta-analysis of multiple studies, which may facilitate the identification of biomarkers to personalize therapy for patients with IBD. The processed single cell count tables are provided in Gene Expression Omnibus; GSE162335. Raw sequence data are not public and are protected by controlled-access for patient privacy., (Copyright © 2021. Published by Elsevier Inc.)

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. The exact oxalate-degrading taxa in the human microbiota and their ODP have not been described. We leverage multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota primarily uses the type II ODP, rather than type I. Furthermore, among the diverse ODP-encoding microbes, an oxalate autotroph, Oxalobacter formigenes , dominates this function transcriptionally. Patients with inflammatory bowel disease (IBD) frequently suffer from disrupted oxalate homeostasis and calcium oxalate nephrolithiasis. We show that the enteric oxalate level is elevated in IBD patients, with highest levels in Crohn's disease (CD) patients with both ileal and colonic involvement consistent with known nephrolithiasis risk. We show that the microbiota ODP expression is reduced in IBD patients, which may contribute to the disrupted oxalate homeostasis. The specific changes in ODP expression by several important taxa suggest that they play distinct roles in IBD-induced nephrolithiasis risk. Lastly, we colonize mice that are maintained in the gnotobiotic facility with O. formigenes , using either a laboratory isolate or an isolate we cultured from human stools, and observed a significant reduction in host fecal and urine oxalate levels, supporting our in silico prediction of the importance of the microbiome, particularly O. formigenes in host oxalate homeostasis., Competing Interests: ML, JD, JH, AV, TB, MH, PL, HL, KR, AT, MB, LN No competing interests declared, JA is an employee of Litholink, AB is an employee of Genentech, (© 2021, Liu et al.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CL pro (M pro ). The drug candidate PF-00835231 is the active compound of the first anti-3CL pro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CL pro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549 +ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549 +ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549 +ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. Importance: The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CL pro (M pro ), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CL pro -targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2., (Copyright © 2021 de Vries et al.)

Staphylococcus aureus is responsible for various diseases in humans, and recurrent infections are commonly observed. S. aureus produces an array of bicomponent pore-forming toxins that target and kill leukocytes, known collectively as the leukocidins. The contribution of these leukocidins to impair the development of anti-S. aureus adaptive immunity and facilitate reinfection is unclear. Using a murine model of recurrent bacteremia, we demonstrate that infection with a leukocidin mutant results in increased levels of anti-S. aureus antibodies compared with mice infected with the WT parental strain, indicating that leukocidins negatively impact the generation of anti-S. aureus antibodies in vivo. We hypothesized that neutralizing leukocidin-mediated immune subversion by vaccination may shift this host-pathogen interaction in favor of the host. Leukocidin-immunized mice produce potent leukocidin-neutralizing antibodies and robust Th1 and Th17 responses, which collectively protect against bloodstream infections. Altogether, these results demonstrate that blocking leukocidin-mediated immune evasion can promote host protection against S. aureus bloodstream infection., Competing Interests: Disclosures: V.J. Torres reported grants from Janssen Biotech Inc. during the conduct of the study outside the submitted work; in addition, V.J. Torres has patents to use leukocidins as S. aureus vaccine antigens, which are licensed to Janssen Biotech Inc. No other disclosures were reported., (© 2020 Tam et al.)

Myeloid cells are a vital component of innate immunity and comprise monocytes, macrophages, dendritic cells, and granulocytes. How myeloid cell lineage affects activation states in response to cytokines remains poorly understood. The cytokine environment and cellular infiltrate during an inflammatory response may contain prognostic features that predict disease outcome. In this study, we analyzed the transcriptional responses of human monocytes, macrophages, dendritic cells, and neutrophils in response to stimulation by IFN-γ, IFN-β, IFN-λ, IL-4, IL-13, and IL-10 cytokines to better understand the heterogeneity of activation states in inflammatory conditions. This generated a myeloid cell-cytokine-specific response matrix that can infer representation of myeloid cells and the cytokine environment they encounter during infection, in tumors and in whole blood. Neutrophils were highly responsive to type 1 and type 2 cytokine stimulation but did not respond to IL-10. We identified transcripts specific to IFN-β stimulation, whereas other IFN signature genes were upregulated by both IFN-γ and IFN-β. When we used our matrix to deconvolute blood profiles from tuberculosis patients, the IFN-β-specific neutrophil signature was reduced in tuberculosis patients with active disease, whereas the shared response to IFN-γ and IFN-β in neutrophils was increased. When applied to glioma patients, transcripts of neutrophils exposed to IL-4/IL-13 and monocyte responses to IFN-γ or IFN-β emerged as opposing predictors of patient survival. Hence, by dissecting how different myeloid cells respond to cytokine activation, we can delineate biological roles for myeloid cells in different cytokine environments during disease processes, especially during infection and tumor progression., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The relative contributions of genetic and environmental factors to variation in immune responses are poorly understood. Here, we performed a phenotypic analysis of immunological parameters in laboratory mice carrying susceptibility genes implicated in inflammatory bowel disease (IBD) (Nod2 and Atg16l1) upon exposure to environmental microbes. Mice were released into an outdoor enclosure (rewilded) and then profiled for immune responses in the blood and lymph nodes. Variations of immune cell populations were largely driven by the environment, whereas cytokine production elicited by microbial antigens was more affected by the genetic mutations. We identified transcriptional signatures in the lymph nodes associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans, and Bacteroides vulgatus were also coupled with rewilding. Therefore, exposing laboratory mice with genetic mutations to a natural environment uncovers different contributions to variations in microbial responses and immune cell composition., Competing Interests: Declaration of Interests K.C. receives research funding from Pfizer and Abbvie, and P.L. receives research funding from Pfizer. K.C. has consulted for or received an honorarium from Puretech Health, Genentech, and Abbvie. P.L. consults for and has equity in Toilabs. K.C. has a provisional patent, U.S. Patent Appln. No. 15/625,934. P.L. is a federal employee. All other authors declare no competing interests., (Published by Elsevier Inc.)

Free-living mammals, such as humans and wild mice, display heightened immune activation compared with artificially maintained laboratory mice. These differences are partially attributed to microbial exposure as laboratory mice infected with pathogens exhibit immune profiles more closely resembling that of free-living animals. Here, we examine how colonization by microorganisms within the natural environment contributes to immune system maturation by releasing inbred laboratory mice into an outdoor enclosure. In addition to enhancing differentiation of T cell populations previously associated with pathogen exposure, outdoor release increased circulating granulocytes. However, these "rewilded" mice were not infected by pathogens previously implicated in immune activation. Rather, immune system changes were associated with altered microbiota composition with notable increases in intestinal fungi. Fungi isolated from rewilded mice were sufficient in increasing circulating granulocytes. These findings establish a model to investigate how the natural environment impacts immune development and show that sustained fungal exposure impacts granulocyte numbers., Competing Interests: Declaration of Interests K.C. receives research funding from Pfizer and Abbvie and P.L. receives research funding from Pfizer. K.C. has consulted for or received an honorarium from Puretech Health, Genentech, and Abbvie. K.C. has a provisional patent, U.S. Patent Application. No. 15/625,934. P.L. consults for and has equity in Toilabs. P.L. is a federal employee., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tissue-resident macrophages are a diverse population of cells that perform specialized functions including sustaining tissue homeostasis and tissue surveillance. Here, we report an interstitial subset of CD169 + lung-resident macrophages that are transcriptionally and developmentally distinct from alveolar macrophages (AMs). They are primarily localized around the airways and are found in close proximity to the sympathetic nerves in the bronchovascular bundle. These nerve- and airway-associated macrophages (NAMs) are tissue resident, yolk sac derived, self-renewing, and do not require CCR2 + monocytes for development or maintenance. Unlike AMs, the development of NAMs requires CSF1 but not GM-CSF. Bulk population and single-cell transcriptome analysis indicated that NAMs are distinct from other lung-resident macrophage subsets and highly express immunoregulatory genes under steady-state and inflammatory conditions. NAMs proliferated robustly after influenza infection and activation with the TLR3 ligand poly(I:C), and in their absence, the inflammatory response was augmented, resulting in excessive production of inflammatory cytokines and innate immune cell infiltration. Overall, our study provides insights into a distinct subset of airway-associated pulmonary macrophages that function to maintain immune and tissue homeostasis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Single-cell RNA sequencing (scRNA-seq) enables the systematic identification of cell populations in a tissue, but characterizing their spatial organization remains challenging. We combine a microarray-based spatial transcriptomics method that reveals spatial patterns of gene expression using an array of spots, each capturing the transcriptomes of multiple adjacent cells, with scRNA-Seq generated from the same sample. To annotate the precise cellular composition of distinct tissue regions, we introduce a method for multimodal intersection analysis. Applying multimodal intersection analysis to primary pancreatic tumors, we find that subpopulations of ductal cells, macrophages, dendritic cells and cancer cells have spatially restricted enrichments, as well as distinct coenrichments with other cell types. Furthermore, we identify colocalization of inflammatory fibroblasts and cancer cells expressing a stress-response gene module. Our approach for mapping the architecture of scRNA-seq-defined subpopulations can be applied to reveal the interactions inherent to complex tissues.

Helminth infection and dietary intake can affect the intestinal microbiota, as well as the immune system. Here we analyzed the relationship between fecal microbiota and blood profiles of indigenous Malaysians, referred to locally as Orang Asli, in comparison to urban participants from the capital city of Malaysia, Kuala Lumpur. We found that helminth infections had a larger effect on gut microbial composition than did dietary intake or blood profiles. Trichuris trichiura infection intensity also had the strongest association with blood transcriptional profiles. By characterizing paired longitudinal samples collected before and after deworming treatment, we determined that changes in serum zinc and iron levels among the Orang Asli were driven by changes in helminth infection status, independent of dietary metal intake. Serum zinc and iron levels were associated with changes in the abundance of several microbial taxa. Hence, there is considerable interplay between helminths, micronutrients and the microbiota on the regulation of immune responses in humans., Competing Interests: The authors have declared that no competing interests exist.

Background: Reproductive aging may impact the vaginal microbiome and genital tract mucosal immune environment and contribute to genital tract health in women living with and at-risk for HIV infection., Methods: A cross-sectional study of 102 HIV+ (51 premenopausal, 51 postmenopausal) and 39 HIV-uninfected (HIV-) (20 premenopausal, 19 postmenopausal) women was performed in Bronx and Brooklyn, NY. Cervicovaginal lavage (CVL) was collected for quantification of innate antimicrobial activity against E. coli, HSV-2 and HIV and immune mediators by Luminex and ELISA. Microbiome studies by qPCR and 16S rRNA sequencing were performed on vaginal swabs., Results: HIV+ postmenopausal compared to premenopausal participants had lower median E. coli bactericidal activity (41% vs. 62%, p = 0.001), lower median gene copies of Lactobacillus crispatus (p = 0.005) and Lactobacillus iners (p = 0.019), lower proportions of Lactobacillus iners, higher proportions of Gardnerella and Atopobium vaginae and lower levels of human beta defensins (HBD-2, HBD-3) and secretory leukocyte protease inhibitor (SLPI), p<0.001. HSV-2 inhibitory activity was higher in HIV+ postmenopausal compared to premenopausal participants (37% vs. 17%, p = 0.001) and correlated with the proinflammatory molecules interleukin (IL) 6, IL-8, human neutrophil peptide (HNP) 1-3, lactoferrin and fibronectin. Similar trends were observed in HIV- postmenopausal compared to premenopausal participants. HIV inhibitory activity did not differ by reproductive status in the HIV+ participants but was significantly higher in HIV- postmenopausal compared to premenopausal participants and in participants with suppressed plasma viral load, and inversely correlated with gene copies of G. vaginalis and BVAB2. A significant proportion of HIV+ participants on ART exhibited HIV enhancing activity., Conclusions: HIV+ postmenopausal compared to premenopausal participants have less CVL E. coli bactericidal activity, reflecting a reduction in Lactobacilli and a greater proportion of Gardnerella and A. vaginae, and more HSV-2 inhibitory activity, reflecting increased mucosal inflammation. The effect of menopause on mucosal immunity was greater in HIV+ participants, suggesting a synergistic impact. Promotion of a lactobacillus dominant vaginal microbiome and reduced mucosal inflammation may improve vaginal health and reduce risk for shedding of HIV and potential for HIV transmission in HIV+ menopausal women., Competing Interests: The authors have declared that no competing interests exist.

Background: Exploration of large data sets, such as shotgun metagenomic sequence or expression data, by biomedical experts and medical professionals remains as a major bottleneck in the scientific discovery process. Although tools for this purpose exist for 16S ribosomal RNA sequencing analysis, there is a growing but still insufficient number of user-friendly interactive visualization workflows for easy data exploration and figure generation. The development of such platforms for this purpose is necessary to accelerate and streamline microbiome laboratory research., Results: We developed the Workflow Hub for Automated Metagenomic Exploration (WHAM!) as a web-based interactive tool capable of user-directed data visualization and statistical analysis of annotated shotgun metagenomic and metatranscriptomic data sets. WHAM! includes exploratory and hypothesis-based gene and taxa search modules for visualizing differences in microbial taxa and gene family expression across experimental groups, and for creating publication quality figures without the need for command line interface or in-house bioinformatics., Conclusions: WHAM! is an interactive and customizable tool for downstream metagenomic and metatranscriptomic analysis providing a user-friendly interface allowing for easy data exploration by microbiome and ecological experts to facilitate discovery in multi-dimensional and large-scale data sets.

Epidemiological evidence supports a direct association between early microbiota impact-including C-section-and obesity. We performed antibiotic-free, fostered C-sections and determined the impact on the early microbiota and body weight during development. Mice in the C-section group gained more body mass after weaning, with a stronger phenotype in females. C-section-born mice lacked the dynamic developmental gut microbiota changes observed in control mice. The results demonstrate a causal relationship between C-section and increased body weight, supporting the involvement of maternal vaginal bacteria in normal metabolic development.

We have studied the pharmacokinetics of cis-trans, trans-trans and cis-cis mivacurium in nine healthy patients (creatinine clearance 66-133 ml min-1), in seven patients with end-stage renal failure requiring dialysis (creatinine clearance 4-11 ml min-1) and in seven patients with impaired renal function (creatinine clearance 32-49 ml min-1), during thiopentone-fentanyl-midazolam-nitrous oxide-oxygen anaesthesia. Mivacurium chloride was infused at a rate of 15 micrograms kg-1 min-1 for 10 min, 7.5 micrograms kg-1 min-1 for a further 10 min, and then at a rate adjusted to maintain T1/T0 at 5%. The minimum duration of infusion was 60 min (range 60-235 min). The plasma concentration of the three isomers was measured at regular intervals throughout the infusion, and for up to 300 min after the infusion was stopped. Compartmental analysis of the resulting isomer profiles was undertaken: one- and two-compartment models were fitted to derive clearance, volume of distribution and terminal elimination half-life. Clearance of the cis-cis isomer was reduced significantly in the renal failure (median 2.4 (range 2.1-2.6) ml kg-1 min-1) and intermediate renal function groups (2.1 (2.0-2.9) ml kg-1 min-1), compared with healthy patients (3.8 (2.6-4.9) ml kg-1 min-1) (P < 0.01 in each case).(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the pharmokinetics of cis-trans, trans-trans and cis-cis mivacurium in 10 healthy subjects and 11 patients with mild or moderate hepatic cirrhosis, during nitrous oxide-oxygen-isoflurane anaesthesia. Mivacurium 15 micrograms kg-1 min-1 was infused for 10 min (total dose 0.15 mg kg-1) and the plasma concentration of the three isomers measured at regular intervals for 190 min. The electromyographic response to the drug was also measured. Compartmental analysis of the resulting isomer profiles was undertaken: one- and two-compartment models were fitted to derive clearance, volume of distribution and half-life. Clearance of the cis-trans and trans-trans isomers was reduced significantly in the cirrhotic compared with the healthy group: cis-trans (median (range)) 44 (15-121) ml kg-1 min-1 vs 95 (57-213) ml kg-1 min-1 (P < 0.05); trans-trans 32 (12-64) ml kg-1 min-1 vs 70 (34-101) ml kg-1 min-1 (P < 0.05). The difference in the clearance of the cis-cis isomer in the cirrhotic (4.2 (2.9-12.1) ml kg-1 min-1) compared with the healthy group (5.2 (2.9-8.9) ml kg-1 min-1) was not significant with this sample size. Clearance of each isomer correlated significantly with plasma cholinesterase activity: cis-trans r = 0.73, P < 0.001; trans-trans r = 0.69, P < 0.001; cis-cis r = 0.48, P < 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)

Ten healthy patients and 25 patients with cirrhosis of the liver (10 Child's A, 10 Child's B and 5 Child's C) received a bolus dose of mivacurium chloride 150 micrograms kg-1. The electromyographic response was monitored throughout anaesthesia until recovery of the first twitch of the train-of-four (TOF) (T1/T0) to at least 85% and the TOF ratio (T4:T1) to at least 80%. There was no significant difference between the two groups in the onset of neuromuscular block, but recovery was prolonged in the cirrhotic group compared with the healthy patients (respective mean times to recovery of T1/T0: to 5% = 20.2 vs 11.2 min (P < 0.05); to 10% = 23.8 vs 13.4 min (P < 0.005); to 25% = 28.4 vs 16.6 min (P < 0.005); to 50% = 41.1 vs 20.1 min (P < 0.005); to 75% = 43.8 vs 24.9 min (P < 0.005). Recovery of T4:T1 to 70% = 48.1 vs 27.4 min (P < 0.005)). Recovery was most prolonged in the Child's C patients. Mean plasma cholinesterase activity was less in the cirrhotic compared with the healthy group (mean 582 (SD 254) iu litre-1 vs 1125 (303) iu litre-1) (P < 0.001) and there was a significant negative correlation between plasma cholinesterase activity and all the indices of recovery (P < 0.001 for all except recovery index (P < 0.01)). We conclude that patients with hepatic cirrhosis may be sensitive to mivacurium, which could be explained, at least in part, by the lesser plasma cholinesterase activity.

1. Whole body protein turnover was measured using a primed-constant infusion of L-[1-13C]leucine with measurement of breath 13CO2 production and plasma 13C alpha-ketoisocaproate enrichment. Ten fasting patients, requiring mechanical ventilation and suffering from multiple organ failure, and six healthy control subjects were studied. 2. Protein breakdown and leucine removal from the plasma for protein synthesis were significantly higher in the patients than in the control subjects (P < 0.01). In addition, leucine oxidation was almost 75% higher in the patients than in the healthy control subjects (P < 0.05). 3. Plasma concentrations of glucose, insulin and growth hormone were not different between the two groups, but those of glucagon (not significant), noradrenaline (P < 0.05) and cortisol (P < 0.01) were almost two- and three-fold higher in the patients than in the control subjects. 4. Mean energy expenditure, measured by indirect calorimetry, was 30% higher in the patients than in the healthy control subjects (P < 0.01). 5. Combining the data from both groups of subjects and using multiple regression analysis, cortisol was found to be the most significant predictor of (i) protein breakdown (48% of variance explained), (ii) leucine oxidation (69%) and (iii) hourly energy expenditure (54%). 6. The present investigation using [13C]leucine tracer methods demonstrated, in patients with multiple organ failure, that whole body protein breakdown and synthesis increased concomitantly and were twice as high as rates measured in healthy control subjects. Of the hormones measured in the present study, cortisol appears to have the most significant effect on whole body protein turnover.