Smith-specific regulatory T cells halt the progression of lupus nephritis.

Antigen-specific regulatory T cells (Tregs) suppress pathogenic autoreactivity and are potential therapeutic candidates for autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis is associated with autoreactivity to the Smith (Sm) autoantigen and the human leucocyte antigen (HLA)-DR15 haplotype; hence, we investigated the potential of Sm-specific Tregs (Sm-Tregs) to suppress disease. Here we identify a HLA-DR15 restricted immunodominant Sm T cell epitope using biophysical affinity binding assays, then identify high-affinity Sm-specific T cell receptors (TCRs) using high-throughput single-cell sequencing. Using lentiviral vectors, we transduce our lead Sm-specific TCR into Tregs derived from patients with SLE who are anti-Sm and HLA-DR15 positive. Compared with polyclonal mock-transduced Tregs, Sm-Tregs potently suppress Sm-specific pro-inflammatory responses in vitro and suppress disease progression in a humanized mouse model of lupus nephritis. These results show that Sm-Tregs are a promising therapy for SLE.

Magnetic Bead-Based Workflow for Sensitive and Streamlined Cell Surface Proteomics.

Cell surface proteins represent an important class of molecules for therapeutic targeting and cellular phenotyping. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, post-translational modifications, hydrophobic regions, and processing requirements. To improve their identification, we optimized a Cell-Surface Capture (CSC) workflow that incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), nonenzymatic deamidation (digestion and deglycosylation buffers), and data acquisition methods (DDA, DIA, and TMT). Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid, SDS/urea-based lysis buffers, single-pot solid-phased-enhanced sample-preparation (SP3), and streptavidin magnetic beads for maximal surfaceome coverage. Notably, with semiautomated processing, sample handling was simplified and between ∼600 and 900 cell surface N-glycoproteins were identified from only 25-200 µg of HeLa protein. CSC also revealed significant differences between in vitro monolayer cultures and in vivo tumor xenografts of murine CT26 colon adenocarcinoma samples that may aid in target identification for drug development. Overall, the improved efficiency of the magnetic-based CSC workflow identified both previously reported and novel N-glycosites with less material and high reproducibility that should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

Imidazolone as an Amide Bioisostere in the Development of ß-1,3-N-Acetylglucosaminyltransferase 2 (B3GNT2) Inhibitors.

B3GNT2 is responsible for elongation of cell surface long-chain polylactosamine, which influences the regulation of the immune response, making it an attractive target for immunomodulation. In the development of amide containing B3GNT2 inhibitors guided by structure-based drug design, imidazolones were found to successfully serve as amide bioisosteres. This novel imidazolone isosteric strategy alleviated torsional strain of the amide bond on binding to B3GNT2 and improved potency, isoform selectivity, as well as certain physicochemical and pharmacokinetic properties. Herein, we present the synthesis, SAR, X-ray cocrystal structures, and in vivo PK properties of imidazol-4-ones in the context of B3GNT2 inhibition.

Enhancing the Prefusion Conformational Stability of SARS-CoV-2 Spike Protein Through Structure-Guided Design.

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.

STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets.

Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/ß sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.

Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling.

BACKGROUND: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. RESULTS: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5' v1 and 3' v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. CONCLUSION: Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

Structures and mechanism of human glycosyltransferase ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), an important player in immune homeostasis.

ß1,3-N-acetylglucosaminyltransferases (B3GNTs) are Golgi-resident glycosyltransferases involved in the biosynthesis of poly-N-acetyl-lactosamine chains. They catalyze the addition of the N-acetylglucosamine to the N-acetyl-lactosamine repeat as a key step of the chain elongation process. Poly-N-acetyl-lactosamine is involved in the immune system in many ways. Particularly, its long chain has been demonstrated to suppress excessive immune responses. Among the characterized B3GNTs, B3GNT2 is the major poly-N-acetyl-lactosamine synthase, and deletion of its coding gene dramatically reduced the cell surface poly-N-acetyl-lactosamine and led to hypersensitive and hyperresponsive immunocytes. Despite the extensive functional studies, no structural information is available to understand the molecular mechanism of B3GNT2, as well as other B3GNTs. Here we present the structural and kinetic studies of the human B3GNT2. Five crystal structures of B3GNT2 have been determined in the unliganded, donor substrate-bound, acceptor substrate-bound, and product(s)-bound states at resolutions ranging from 1.85 to 2.35 Å. Kinetic study shows that the transglycosylation reaction follows a sequential mechanism. Critical residues involved in recognition of both donor and acceptor substrates as well as catalysis are identified. Mutations of these invariant residues impair B3GNT2 activity in cell assays. Structural comparison with other glycosyltransferases such as mouse Fringe reveals a novel N-terminal helical domain of B3GNTs that may stabilize the catalytic domain and distinguish among different acceptor substrates.

Neutrophil serine protease 4 is required for mast cell-dependent vascular leakage.

Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.

Correction: Crystal structures of human procathepsin H.

[This corrects the article DOI: 10.1371/journal.pone.0200374.].

A homozygous loss-of-function mutation leading to CYBC1 deficiency causes chronic granulomatous disease.

Mutations in genes encoding subunits of the phagocyte NADPH oxidase complex are recognized to cause chronic granulomatous disease (CGD), a severe primary immunodeficiency. Here we describe how deficiency of CYBC1, a previously uncharacterized protein in humans (C17orf62), leads to reduced expression of NADPH oxidase's main subunit (gp91phox) and results in CGD. Analyzing two brothers diagnosed with CGD we identify a homozygous loss-of-function mutation, p.Tyr2Ter, in CYBC1. Imputation of p.Tyr2Ter into 155K chip-genotyped Icelanders reveals six additional homozygotes, all with signs of CGD, manifesting as colitis, rare infections, or a severely impaired PMA-induced neutrophil oxidative burst. Homozygosity for p.Tyr2Ter consequently associates with inflammatory bowel disease (IBD) in Iceland (P = 8.3 × 10-8; OR = 67.6), as well as reduced height (P = 3.3 × 10-4; -8.5 cm). Overall, we find that CYBC1 deficiency results in CGD characterized by colitis and a distinct profile of infections indicative of macrophage dysfunction.

Crystal structures of human procathepsin H.

Cathepsin H is a member of the papain superfamily of lysosomal cysteine proteases. It is the only known aminopeptidase in the family and is reported to be involved in cancer and other major diseases. Like many other proteases, it is synthesized as an inactive proenzyme. Although the crystal structure of mature porcine cathepsin H revealed the binding of the mini-chain and provided structural basis for the aminopeptidase activity, detailed structural and functional information on the inhibition and activation of procathepsin H has been elusive. Here we present the crystal structures of human procathepsin H at 2.00 Å and 1.66 Å resolution. These structures allow us to explore in detail the molecular basis for the inhibition of the mature domain by the prodomain. Comparison with cathepsin H structure reveals how mini-chain reorients upon activation. We further demonstrate that procathepsin H is not auto-activated but can be trans-activated by cathepsin L.

Inflammatory Bowel Disease Susceptibility Gene C1ORF106 Regulates Intestinal Epithelial Permeability.

Intestinal epithelial cells form a physical barrier that is tightly regulated to control intestinal permeability. Proinflammatory cytokines, such as TNF-α, increase epithelial permeability through disruption of epithelial junctions. The regulation of the epithelial barrier in inflammatory gastrointestinal disease remains to be fully characterized. In this article, we show that the human inflammatory bowel disease genetic susceptibility gene C1ORF106 plays a key role in regulating gut epithelial permeability. C1ORF106 directly interacts with cytohesins to maintain functional epithelial cell junctions. C1orf106-deficient mice are hypersensitive to TNF-α-induced increase in epithelial permeability, and this is associated with increased diarrhea. This study identifies C1ORF106 as an epithelial cell junction protein, and the loss of C1ORF106 augments TNF-α-induced intestinal epithelial leakage and diarrhea that may play a critical role in the development of inflammatory bowel disease.

Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells.

The emergence of Mycobacterium tuberculosis (MTB) strains that are resistant to most or all available antibiotics has created a severe problem for treating tuberculosis and has spurred a quest for new antibiotic targets. Here, we demonstrate that trans-translation is essential for growth of MTB and is a viable target for development of antituberculosis drugs. We also show that an inhibitor of trans-translation, KKL-35, is bactericidal against MTB under both aerobic and anoxic conditions. Biochemical experiments show that this compound targets helix 89 of the 23S rRNA. In silico molecular docking predicts a binding pocket for KKL-35 adjacent to the peptidyl-transfer center in a region not targeted by conventional antibiotics. Computational solvent mapping suggests that this pocket is a druggable hot spot for small molecule binding. Collectively, our findings reveal a new target for antituberculosis drug development and provide critical insight on the mechanism of antibacterial action for KKL-35 and related 1,3,4-oxadiazole benzamides.

Mice deficient in NRROS show abnormal microglial development and neurological disorders.

Microglia and other tissue-resident macrophages within the central nervous system (CNS) have essential roles in neural development, inflammation and homeostasis. However, the molecular pathways underlying their development and function remain poorly understood. Here we report that mice deficient in NRROS, a myeloid-expressed transmembrane protein in the endoplasmic reticulum, develop spontaneous neurological disorders. NRROS-deficient (Nrros-/-) mice show defects in motor functions and die before 6 months of age. Nrros-/- mice display astrogliosis and lack normal CD11bhiCD45lo microglia, but they show no detectable demyelination or neuronal loss. Instead, perivascular macrophage-like myeloid cells populate the Nrros-/- CNS. Cx3cr1-driven deletion of Nrros shows its crucial role in microglial establishment during early embryonic stages. NRROS is required for normal expression of Sall1 and other microglial genes that are important for microglial development and function. Our study reveals a NRROS-mediated pathway that controls CNS-resident macrophage development and affects neurological function.

Deciphering the crosstalk among IL-1 and IL-10 family cytokines in intestinal immunity.

The IL-1 and IL-10 family cytokines are important regulators of intestinal immunity. Whereas these cytokines have protective roles in response to mucosal damage or infection, they also contribute to pathology in certain settings. How these cytokines function to maintain intestinal homoeostasis, and under what circumstances they contribute to disease is poorly understood. Recent studies have revealed a multi-layered regulatory network wherein IL-1 and IL-10 family cytokines impact each other's production. The workings of this network vary in different intestinal regions, reflecting the influence of resident microbiota and the distribution of distinct immune cell populations in different regions of the intestine. We review these findings here, and discuss them in the context of the current understanding of the functions of these cytokine families in health and disease. We further highlight important areas of future investigation.

Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.

T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T. gondii or Citrobacter rodentium infection, but also maintained the homeostatic amount of proIL-18 in the ileum. IL-22, however, did not induce the processing to active IL-18, suggesting a two-step regulation of IL-18 in these cells. Although IL-18 exerted pathogenic functions during ileitis triggered by T. gondii, it was required for host defense against C. rodentium. Conversely, IL-18 was required for the expression of IL-22 in innate lymphoid cells (ILCs) upon T. gondii infection. Our results define IL-18 as an IL-22 target gene in epithelial cells and describe a complex mutual regulation of both cytokines during intestinal infection.

Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes.

The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine. Here we investigate the connection between IL-22 and metabolic disorders. We find that the induction of IL-22 from innate lymphoid cells and CD4(+) T cells is impaired in obese mice under various immune challenges, especially in the colon during infection with Citrobacter rodentium. While innate lymphoid cell populations are largely intact in obese mice, the upregulation of IL-23, a cytokine upstream of IL-22, is compromised during the infection. Consequently, these mice are susceptible to C. rodentium infection, and both exogenous IL-22 and IL-23 are able to restore the mucosal host defence. Importantly, we further unveil unexpected functions of IL-22 in regulating metabolism. Mice deficient in IL-22 receptor and fed with high-fat diet are prone to developing metabolic disorders. Strikingly, administration of exogenous IL-22 in genetically obese leptin-receptor-deficient (db/db) mice and mice fed with high-fat diet reverses many of the metabolic symptoms, including hyperglycaemia and insulin resistance. IL-22 shows diverse metabolic benefits, as it improves insulin sensitivity, preserves gut mucosal barrier and endocrine functions, decreases endotoxaemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. In summary, we identify the IL-22 pathway as a novel target for therapeutic intervention in metabolic diseases.

cor, a novel carbon monoxide resistance gene, is essential for Mycobacterium tuberculosis pathogenesis.

UNLABELLED: Tuberculosis, caused by Mycobacterium tuberculosis, remains a devastating human infectious disease, causing two million deaths annually. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, Cor) leads to marked CO sensitivity. Heterologous expression of Cor in Escherichia coli rescued it from CO toxicity. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism. IMPORTANCE: Macrophages produce a variety of antimicrobial molecules, including nitric oxide (NO), hydrogen peroxide (H2O2), and acid (H+), that serve to kill engulfed bacteria. In addition to these molecules, human and mouse macrophages also produce carbon monoxide (CO) gas by the heme oxygenase (HO1) enzyme. We observed that, in contrast to other bacteria, mycobacteria are resistant to CO, suggesting that this might be an evolutionary adaptation of mycobacteria for survival within macrophages. We screened a panel of ~2,500 M. tuberculosis mutants to determine which genes are required for survival of M. tuberculosis in the presence of CO. Within this panel, we identified one such gene, cor, that specifically confers CO resistance. Importantly, we found that the ability of M. tuberculosis cells carrying a mutated copy of this gene to cause tuberculosis in a mouse disease model is significantly attenuated. This indicates that CO resistance is essential for mycobacterial survival in vivo.

The ubiquitin ligase parkin mediates resistance to intracellular pathogens.

Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson's disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

Salmonella require the fatty acid regulator PPARδ for the establishment of a metabolic environment essential for long-term persistence.

Host-adapted Salmonella strains are responsible for a number of disease manifestations in mammals, including an asymptomatic chronic infection in which bacteria survive within macrophages located in systemic sites. However, the host cell physiology and metabolic requirements supporting bacterial persistence are poorly understood. In a mouse model of long-term infection, we found that S. typhimurium preferentially associates with anti-inflammatory/M2 macrophages at later stages of infection. Further, PPARδ, a eukaryotic transcription factor involved in sustaining fatty acid metabolism, is upregulated in Salmonella-infected macrophages. PPARδ deficiency dramatically inhibits Salmonella replication, which is linked to the metabolic state of macrophages and the level of intracellular glucose available to bacteria. Pharmacological activation of PPARδ increases glucose availability and enhances bacterial replication in macrophages and mice, while Salmonella fail to persist in Pparδ null mice. These data suggest that M2 macrophages represent a unique niche for long-term intracellular bacterial survival and link the PPARδ-regulated metabolic state of the host cell to persistent bacterial infection.