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Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites. [ABSTRACT FROM AUTHOR]

The nucleotide-binding-domain (NBD)–and leucine-rich-repeat (LRR)–containing (NLR) family, pyrin-domain–containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the domain conserved in NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants. [ABSTRACT FROM AUTHOR]

Lymphoid T-zone fibroblastic reticular cells (FRCs) actively promote T-cell trafficking, homeostasis, and expansion but can also attenuate excessive T-cell responses via inducible nitric oxide (NO) and constitutive prostanoid release. It remains unclear how these FRC-derived mediators dampen T-cell responses and whether this occurs in vivo. Here, we confirm that murine lymph node (LN) FRCs produce prostaglandin E2 (PGE2) in a cyclooxygenase-2 (COX2)-dependent and inflammation-independent fashion. We show that this COX2/PGE2 pathway is active during both strong and weak T-cell responses, in contrast to NO, which only comes into play during strong T-cell responses. During chronic infections in vivo, PGE2-receptor signaling in virus-specific cluster of differentiation (CD)8 cytotoxic T cells was shown by others to suppress T-cell survival and function. Using COX2flox/flox mice crossed to mice expressing Cre recombinase expression under control of the CC chemokine ligand (CCL19) promoter (CCL19cre), we now identify CCL19+ FRC as the critical source of this COX2-dependent suppressive factor, suggesting PGE2-expressing FRCs within lymphoid tissues are an interesting therapeutic target to improve T-cell–mediated pathogen control during chronic infection. [ABSTRACT FROM AUTHOR]

The assessment of the regenerative capacity of the heart has been compromised by the lack of surface signatures to characterize cardiomyocytes (CMs). Here, combined multiparametric surface marker analysis with single-cell transcriptional profiling and in vivo transplantation identify the main mouse fetal cardiac populations and their progenitors (PRGs). We found that CMs at different stages of differentiation coexist during development. We identified a population of immature heat stable antigen (HSA)/ cluster of differentiation 24 (CD24)+ CMs that persists throughout life and that, unlike other CM subsets, actively proliferates up to 1 week of age and engrafts cardiac tissue upon transplantation. In the adult heart, a discrete population of HSA/CD24+ CMs appears as mononucleated cells that increase in frequency after infarction. Our work identified cell surface signatures that allow the prospective isolation of CMs at all developmental stages and the detection of a subset of immature CMs throughout life that, although at reduced frequencies, are poised for activation in response to ischemic stimuli. This work opens new perspectives in the understanding and treatment of heart pathologies. [ABSTRACT FROM AUTHOR]

Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with “Fc-unmodified” chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 μg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) “complement-inactive” Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q−/− mice, when C5 function was blocked, or in C9−/− mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics. [ABSTRACT FROM AUTHOR]

Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic “star” nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes. [ABSTRACT FROM AUTHOR]

During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM. [ABSTRACT FROM AUTHOR]

Regulatory T (Treg) cells play central roles in maintaining immune homeostasis and self-tolerance. However, the molecular mechanisms underlying Treg cell homeostasis and suppressive function are still not fully understood. Here, we report that the deletion of another P subfamily members of the forkhead box (Foxp) subfamily member Foxp1 in Treg cells led to increased numbers of activated Treg (aTreg) cells at the expense of quiescent Treg cells, and also resulted in impaired Treg suppressive function. Mice with Foxp1-deficient Treg cells developed spontaneous inflammatory disease with age; they also had more severe inflammatory disease in colitis and experimental autoimmune encephalomyelitis (EAE) models. Mechanistically, we found that Foxp1 bound to the conserved noncoding sequence 2 (CNS2) element of the Foxp3 locus and helped maintain Treg suppressive function by stabilizing the Foxp3 expression. Furthermore, we found that Foxp1 and Foxp3 coordinated the regulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression levels. Taken together, our study demonstrates that Foxp1 plays critical roles in both maintaining Treg cell quiescence during homeostasis and regulating Treg suppressive function. [ABSTRACT FROM AUTHOR]

Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (Rbpj) or Notch1 and Notch2 in T cells. Rbpj-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. Rbpj-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in Rbpj-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in Rbpj-deficient TCRαβ+CD8αα+ IELs acts as an “eat-me” signal. Together, these results revealed that Notch–Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs. [ABSTRACT FROM AUTHOR]

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa. [ABSTRACT FROM AUTHOR]

Hepatitis A virus (HAV), an enigmatic and ancient pathogen, is a major causative agent of acute viral hepatitis worldwide. Although there are effective vaccines, antivirals against HAV infection are still required, especially during fulminant hepatitis outbreaks. A more in-depth understanding of the antigenic characteristics of HAV and the mechanisms of neutralization could aid in the development of rationally designed antiviral drugs targeting HAV. In this paper, 4 new antibodies—F4, F6, F7, and F9—are reported that potently neutralize HAV at 50% neutralizing concentration values (neut50) ranging from 0.1 nM to 0.85 nM. High-resolution cryo-electron microscopy (cryo-EM) structures of HAV bound to F4, F6, F7, and F9, together with results of our previous studies on R10 fragment of antigen binding (Fab)-HAV complex, shed light on the locations and nature of the epitopes recognized by the 5 neutralizing monoclonal antibodies (NAbs). All the epitopes locate within the same patch and are highly conserved. The key structure-activity correlates based on the antigenic sites have been established. Based on the structural data of the single conserved antigenic site and key structure-activity correlates, one promising drug candidate named golvatinib was identified by in silico docking studies. Cell-based antiviral assays confirmed that golvatinib is capable of blocking HAV infection effectively with a 50% inhibitory concentration (IC50) of approximately 1 μM. These results suggest that the single conserved antigenic site from complete HAV capsid is a good antiviral target and that golvatinib could function as a lead compound for anti-HAV drug development. [ABSTRACT FROM AUTHOR]

Gram+ infections are worldwide life-threatening diseases in which the pathological role of type I interferon (IFN) has been highlighted. Plasmacytoid predendritic cells (pDCs) produce high amounts of type I IFN following viral sensing. Despite studies suggesting that pDCs respond to bacteria, the mechanisms underlying bacterial sensing in pDCs are unknown. We show here that human primary pDCs express toll-like receptor 1 (TLR1) and 2 (TLR2) and respond to bacterial lipoproteins. We demonstrated that pDCs differentially respond to gram+ bacteria through the TLR1/2 pathway. Notably, up-regulation of costimulatory molecules and pro-inflammatory cytokines was TLR1 dependent, whereas type I IFN secretion was TLR2 dependent. Mechanistically, we demonstrated that these differences relied on diverse signaling pathways activated by TLR1/2. MAPK and NF-κB pathways were engaged by TLR1, whereas the Phosphoinositide 3-kinase (PI3K) pathway was activated by TLR2. This dichotomy was reflected in a different role of TLR2 and TLR1 in pDC priming of naïve cluster of differentiation 4+ (CD4+) T cells, and T helper (Th) cell differentiation. This work provides the rationale to explore and target pDCs in bacterial infection. [ABSTRACT FROM AUTHOR]

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)–dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides—namely monosialoganglioside GM3 and disialoganglioside GD3—as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells. [ABSTRACT FROM AUTHOR]

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies. [ABSTRACT FROM AUTHOR]

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The “breathing” of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously. [ABSTRACT FROM AUTHOR]

During the entry process, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer undergoes a sequence of conformational changes triggered by both CD4 and coreceptor engagement. Resolving the conformation of these transient entry intermediates has proven challenging. Here, we fine-mapped the antigenicity of entry intermediates induced by increasing CD4 engagement of cell surface–expressed Env. Escalating CD4 triggering led to the sequential adoption of different pre-fusion conformational states of the Env trimer, up to the pre-hairpin conformation, that we assessed for antibody epitope presentation. Maximal accessibility of the coreceptor binding site was detected below Env saturation by CD4. Exposure of the fusion peptide and heptad repeat 1 (HR1) required higher CD4 occupancy. Analyzing the diverse antigenic states of the Env trimer, we obtained key insights into the transitions in epitope accessibility of broadly neutralizing antibodies (bnAbs). Several bnAbs preferentially bound CD4-triggered Env, indicating a potential capacity to neutralize both pre- and post-CD4 engagement, which needs to be explored. Assessing binding and neutralization activity of bnAbs, we confirm antibody dissociation rates as a driver of incomplete neutralization. Collectively, our findings highlight a need to resolve Env conformations that are neutralization-relevant to provide guidance for immunogen development. [ABSTRACT FROM AUTHOR]

Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- β, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-β compared to CD71+VISTA− erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells—compared to CD71+VISTA− and CD71+ erythroid cells from the VISTA KO mice—significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-β in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-β1, TGF-β2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond. [ABSTRACT FROM AUTHOR]

Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which—upon activation by the inflammasomes—translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice. [ABSTRACT FROM AUTHOR]

Forkhead box P3 (Foxp3+) regulatory T (Treg)-cell function is controlled by environmental cues of which cytokine-mediated signaling is a dominant component. In vivo, interleukin-4 (IL-4)-mediated signaling via IL-4 receptor alpha (IL-4Rα) mediates Treg cell transdifferentiation into ex-Foxp3 T helper 2 (Th2) or T helper 17 (Th17) cells. However, IL-4-mediated signaling also reinforces the Foxp3 Treg compartment in vitro. We generated Foxp3-specific IL-4Rα-deficient mice and demonstrated differential efficiency of IL-4Rα deletion in male (approximately 90%) and female (approximately 40%) animals, because of cyclic recombinase (Cre)-mediated X-linked foxp3 inactivation. Irrespective of the degree of IL-4Rα deletion within the Foxp3+ Treg cell population, mice showed exacerbation of immune effector responses with aggravated tissue pathology in tissue-dwelling helminth infections (Schistosoma mansoni or Nippostrongylus brasiliensis). Mechanistically, IL-4Rα deletion in males and females led to a reduced expression of Foxp3 and subsequently an impaired accumulation of Foxp3+ Treg cells to inflamed tissues. In-depth cellular typing by flow cytometry revealed that the impairment of IL-4Rα-mediated signaling during helminth infections decreased the ability of central Treg cells to convert into effector Treg (eTreg) cells and caused a significant down-regulation of markers associated with Treg cell migration (C-X-C motif chemokine receptor 3 [CXCR3]) and accumulation in inflamed tissues (GATA binding protein 3 [GATA3]) as well as survival (B cell lymphoma 2 [Bcl-2]). These findings unprecedentedly, to our knowledge, uncover a role for IL-4Rα signaling in the positive regulation of Foxp3+ Treg cell function in vivo. Complementing our past knowledge on a widely reported role for IL-4Rα signaling in the negative regulation and transdifferentiation of Foxp3+ Treg cells in vivo, our present findings reveal the host requirement for an intact, but not reduced or potentiated, IL-4Rα-mediated signaling on Foxp3+ Treg cells to optimally control inflammation during helminth infections. [ABSTRACT FROM AUTHOR]

Determining the functions of human genes is a key objective for understanding disease and enabling development of new therapeutic approaches. A number of recent studies have shown that the amount of attention the research community gives to each of the more than 20,000 human genes is dramatically skewed toward specific, well-known genes. In this issue, Stoeger and colleagues uncover the factors that explain this bias and offer a way ahead to move more genes into the research limelight. [ABSTRACT FROM AUTHOR]

Leptin is a well-known adipokine that plays a critical role in immune responses. To further explore the immunological roles of leptin, we developed a transgenic leptin pig controlled by the pig leptin (pleptin) promoter to overexpress leptin. Symptoms typically associated with systemic lupus erythematosus (SLE) were evident in this transgenic pig strain, including anemia, leukopenia, and thrombocytopenia as well as kidney and liver impairment. Histologically, there were increased immunoglobulin G (IgG) levels, elevated antiplatelet antibody (APA) levels, and deposition of immune complexes in the kidney and liver. In addition, anti-double-stranded DNA antibodies (dsDNAs), antinuclear antibodies (ANAs), and antinucleosome antibodies (ANuAs) were all significantly increased in serum immunological examinations. These findings were also accompanied by repression of the regulatory T cell (Treg) ratio. Significantly, glucocorticoid experimental therapies partially relieved the autoimmune responses and bleeding symptoms observed in these transgenic leptin pigs. Together, these results indicate that leptin plays a critical role in the development of autoimmune disorders and demonstrate that our transgenic leptin pigs can act as a valuable model of SLE. [ABSTRACT FROM AUTHOR]

Human immunity influences the evolution and impact of influenza strains. Because individuals are infected with multiple influenza strains during their lifetime, and each virus can generate a cross-reactive antibody response, it is challenging to quantify the processes that shape observed immune responses or to reliably detect recent infection from serological samples. Using a Bayesian model of antibody dynamics at multiple timescales, we explain complex cross-reactive antibody landscapes by inferring participants’ histories of infection with serological data from cross-sectional and longitudinal studies of influenza A/H3N2 in southern China and Vietnam. We find that individual-level influenza antibody profiles can be explained by a short-lived, broadly cross-reactive response that decays within a year to leave a smaller long-term response acting against a narrower range of strains. We also demonstrate that accounting for dynamic immune responses alongside infection history can provide a more accurate alternative to traditional definitions of seroconversion for the estimation of infection attack rates. Our work provides a general model for quantifying aspects of influenza immunity acting at multiple timescales based on contemporary serological data and suggests a two-armed immune response to influenza infection consistent with competitive dynamics between B cell populations. This approach to analysing multiple timescales for antigenic responses could also be applied to other multistrain pathogens such as dengue and related flaviviruses. [ABSTRACT FROM AUTHOR]

Determining the duration of protective immunity requires quantifying the magnitude and rate of loss of antibodies to different virus and vaccine antigens. A key complication is heterogeneity in both the magnitude and decay rate of responses of different individuals to a given vaccine, as well as of a given individual to different vaccines. We analyzed longitudinal data on antibody titers in 45 individuals to characterize the extent of this heterogeneity and used models to determine how it affected the longevity of protective immunity to measles, rubella, vaccinia, tetanus, and diphtheria. Our analysis showed that the magnitude of responses in different individuals varied between 12- and 200-fold (95% coverage) depending on the antigen. Heterogeneity in the magnitude and decay rate contribute comparably to variation in the longevity of protective immunity between different individuals. We found that some individuals have, on average, slightly longer-lasting memory than others—on average, they have higher antibody levels with slower decay rates. We identified different patterns for the loss of protective levels of antibodies to different vaccine and virus antigens. Specifically, we found that for the first 25 to 50 years, virtually all individuals have protective antibody titers against diphtheria and tetanus, respectively, but about 10% of the population subsequently lose protective immunity per decade. In contrast, at the outset, not all individuals had protective titers against measles, rubella, and vaccinia. However, these antibody titers wane much more slowly, with a loss of protective immunity in only 1% to 3% of the population per decade. Our results highlight the importance of long-term longitudinal studies for estimating the duration of protective immunity and suggest both how vaccines might be improved and how boosting schedules might be reevaluated. [ABSTRACT FROM AUTHOR]

Fast-moving, competitive fields often inadvertently duplicate research. In a research environment that values being first over being robust, this results in one manuscript “scooping” ongoing research from other groups. Opportunities to demonstrate the solidity of a result through coincidental reproduction are thus lost. Here, two group leaders, one the scooper and one the scoopee, discuss their experiences under PLOS Biology’s new “complementary research” policy. In this case, submission of the second article followed publication of the first by mere days. Scooper and scoopee discuss how complementary research is good for everyone by expanding the scientific reach of studies that are overlapping but not identical, demonstrating the robustness of related results, increasing readership for both authors, and making “replication” studies cost effective by creatively using resources that have already been spent. [ABSTRACT FROM AUTHOR]

Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11–amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of β subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKβ and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKβ and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases. [ABSTRACT FROM AUTHOR]

The oral polio vaccine (OPV) contains live-attenuated polioviruses that induce immunity by causing low virulence infections in vaccine recipients and their close contacts. Widespread immunization with OPV has reduced the annual global burden of paralytic poliomyelitis by a factor of 10,000 or more and has driven wild poliovirus (WPV) to the brink of eradication. However, in instances that have so far been rare, OPV can paralyze vaccine recipients and generate vaccine-derived polio outbreaks. To complete polio eradication, OPV use should eventually cease, but doing so will leave a growing population fully susceptible to infection. If poliovirus is reintroduced after OPV cessation, under what conditions will OPV vaccination be required to interrupt transmission? Can conditions exist in which OPV and WPV reintroduction present similar risks of transmission? To answer these questions, we built a multi-scale mathematical model of infection and transmission calibrated to data from clinical trials and field epidemiology studies. At the within-host level, the model describes the effects of vaccination and waning immunity on shedding and oral susceptibility to infection. At the between-host level, the model emulates the interaction of shedding and oral susceptibility with sanitation and person-to-person contact patterns to determine the transmission rate in communities. Our results show that inactivated polio vaccine (IPV) is sufficient to prevent outbreaks in low transmission rate settings and that OPV can be reintroduced and withdrawn as needed in moderate transmission rate settings. However, in high transmission rate settings, the conditions that support vaccine-derived outbreaks have only been rare because population immunity has been high. Absent population immunity, the Sabin strains from OPV will be nearly as capable of causing outbreaks as WPV. If post-cessation outbreak responses are followed by new vaccine-derived outbreaks, strategies to restore population immunity will be required to ensure the stability of polio eradication. [ABSTRACT FROM AUTHOR]

The immune state of wild animals is largely unknown. Knowing this and what affects it is important in understanding how infection and disease affects wild animals. The immune state of wild animals is also important in understanding the biology of their pathogens, which is directly relevant to explaining pathogen spillover among species, including to humans. The paucity of knowledge about wild animals' immune state is in stark contrast to our exquisitely detailed understanding of the immunobiology of laboratory animals. Making an immune response is costly, and many factors (such as age, sex, infection status, and body condition) have individually been shown to constrain or promote immune responses. But, whether or not these factors affect immune responses and immune state in wild animals, their relative importance, and how they interact (or do not) are unknown. Here, we have investigated the immune ecology of wild house mice—the same species as the laboratory mouse—as an example of a wild mammal, characterising their adaptive humoral, adaptive cellular, and innate immune state. Firstly, we show how immune variation is structured among mouse populations, finding that there can be extensive immune discordance among neighbouring populations. Secondly, we identify the principal factors that underlie the immunological differences among mice, showing that body condition promotes and age constrains individuals’ immune state, while factors such as microparasite infection and season are comparatively unimportant. By applying a multifactorial analysis to an immune system-wide analysis, our results bring a new and unified understanding of the immunobiology of a wild mammal. [ABSTRACT FROM AUTHOR]

Genetic and environmental factors shape host susceptibility to infection, but how and how rapidly environmental variation might alter the susceptibility of mammalian genotypes remains unknown. Here, we investigate the impacts of seminatural environments upon the nematode susceptibility profiles of inbred C57BL/6 mice. We hypothesized that natural exposure to microbes might directly (e.g., via trophic interactions) or indirectly (e.g., via microbe-induced immune responses) alter the hatching, growth, and survival of nematodes in mice housed outdoors. We found that while C57BL/6 mice are resistant to high doses of nematode (Trichuris muris) eggs under clean laboratory conditions, exposure to outdoor environments significantly increased their susceptibility to infection, as evidenced by increased worm burdens and worm biomass. Indeed, mice kept outdoors harbored as many worms as signal transducer and activator of transcription 6 (STAT6) knockout mice, which are genetically deficient in the type 2 immune response essential for clearing nematodes. Using 16S ribosomal RNA sequencing of fecal samples, we discovered enhanced microbial diversity and specific bacterial taxa predictive of nematode burden in outdoor mice. We also observed decreased type 2 and increased type 1 immune responses in lamina propria and mesenteric lymph node (MLN) cells from infected mice residing outdoors. Importantly, in our experimental design, different groups of mice received nematode eggs either before or after moving outdoors. This contrasting timing of rewilding revealed that enhanced hatching of worms was not sufficient to explain the increased worm burdens; instead, microbial enhancement and type 1 immune facilitation of worm growth and survival, as hypothesized, were also necessary to explain our results. These findings demonstrate that environment can rapidly and significantly shape gut microbial communities and mucosal responses to nematode infections, leading to variation in parasite expulsion rates among genetically similar hosts. [ABSTRACT FROM AUTHOR]

Chronic pain is a debilitating problem, and insights in the neurobiology of chronic pain are needed for the development of novel pain therapies. A genome-wide association study implicated the 5p15.2 region in chronic widespread pain. This region includes the coding region for FAM173B, a functionally uncharacterized protein. We demonstrate here that FAM173B is a mitochondrial lysine methyltransferase that promotes chronic pain. Knockdown and sensory neuron overexpression strategies showed that FAM173B is involved in persistent inflammatory and neuropathic pain via a pathway dependent on its methyltransferase activity. FAM173B methyltransferase activity in sensory neurons hyperpolarized mitochondria and promoted macrophage/microglia activation through a reactive oxygen species–dependent pathway. In summary, we uncover a role for methyltransferase activity of FAM173B in the neurobiology of pain. These results also highlight FAM173B methyltransferase activity as a potential therapeutic target to treat debilitating chronic pain conditions. [ABSTRACT FROM AUTHOR]

Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions. [ABSTRACT FROM AUTHOR]

Fundamental to biological decision-making is the ability to generate bimodal expression patterns where 2 alternate expression states simultaneously exist. Here, we use a combination of single-cell analysis and mathematical modeling to examine the sources of bimodality in the transcriptional program controlling HIV’s fate decision between active replication and viral latency. We find that the HIV transactivator of transcription (Tat) protein manipulates the intrinsic toggling of HIV’s promoter, the long terminal repeat (LTR), to generate bimodal ON-OFF expression and that transcriptional positive feedback from Tat shifts and expands the regime of LTR bimodality. This result holds for both minimal synthetic viral circuits and full-length virus. Strikingly, computational analysis indicates that the Tat circuit’s noncooperative “nonlatching” feedback architecture is optimized to slow the promoter’s toggling and generate bimodality by stochastic extinction of Tat. In contrast to the standard Poisson model, theory and experiment show that nonlatching positive feedback substantially dampens the inverse noise-mean relationship to maintain stochastic bimodality despite increasing mean expression levels. Given the rapid evolution of HIV, the presence of a circuit optimized to robustly generate bimodal expression appears consistent with the hypothesis that HIV’s decision between active replication and latency provides a viral fitness advantage. More broadly, the results suggest that positive-feedback circuits may have evolved not only for signal amplification but also for robustly generating bimodality by decoupling expression fluctuations (noise) from mean expression levels. [ABSTRACT FROM AUTHOR]

The bacterium Staphylococcus aureus is an important cause of the life-threatening condition toxic shock syndrome in humans. Bacterial toxins known as superantigens (SAgs) generate this illness by acting as broad activators of a substantial fraction of all T lymphocytes, bypassing the normally highly stringent T-cell receptor antigen specificity to cause a systemic inflammatory cytokine storm in the host. In a new study, Shaler et al. found that immune cells called mucosa-associated invariant T (MAIT) cells make an unexpectedly large contribution to the SAg response in a largely T-cell receptor–independent, cytokine-driven manner. Subsequent to such activation, the MAIT cells remain unresponsive to stimulation with bacterial antigen. Thus, S. aureus hijacks MAIT cells in the cytokine storm and leaves them functionally impaired. This work provides new insight into the role of MAIT cells in antibacterial immunity and opens new avenues of investigation to understand and possibly treat bacterial toxic shock and sepsis. [ABSTRACT FROM AUTHOR]

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a “cytokine storm” with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm’s initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host’s ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ–specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses. [ABSTRACT FROM AUTHOR]

The envelope glycoproteins (Envs) of HIV-1 continuously evolve in the host by random mutations and recombination events. The resulting diversity of Env variants circulating in the population and their continuing diversification process limit the efficacy of AIDS vaccines. We examined the historic changes in Env sequence and structural features (measured by integrity of epitopes on the Env trimer) in a geographically defined population in the United States. As expected, many Env features were relatively conserved during the 1980s. From this state, some features diversified whereas others remained conserved across the years. We sought to identify “clues” to predict the observed historic diversification patterns. Comparison of viruses that cocirculate in patients at any given time revealed that each feature of Env (sequence or structural) exists at a defined level of variance. The in-host variance of each feature is highly conserved among individuals but can vary between different HIV-1 clades. We designate this property “volatility” and apply it to model evolution of features as a linear diffusion process that progresses with increasing genetic distance. Volatilities of different features are highly correlated with their divergence in longitudinally monitored patients. Volatilities of features also correlate highly with their population-level diversification. Using volatility indices measured from a small number of patient samples, we accurately predict the population diversity that developed for each feature over the course of 30 years. Amino acid variants that evolved at key antigenic sites are also predicted well. Therefore, small “fluctuations” in feature values measured in isolated patient samples accurately describe their potential for population-level diversification. These tools will likely contribute to the design of population-targeted AIDS vaccines by effectively capturing the diversity of currently circulating strains and addressing properties of variants expected to appear in the future. [ABSTRACT FROM AUTHOR]

STAT2 is the quintessential transcription factor for type 1 interferons (IFNs), where it functions as a heterodimer with STAT1. However, the human and murine STAT2-deficient phenotypes suggest important additional and currently unidentified type 1 IFN-independent activities. Here, we show that STAT2 constitutively bound to STAT1, but not STAT3, via a conserved interface. While this interaction was irrelevant for type 1 interferon signaling and STAT1 activation, it precluded the nuclear translocation specifically of STAT1 in response to IFN-γ, interleukin-6 (IL-6), and IL-27. This is explained by the dimerization between activated STAT1 and unphosphorylated STAT2, whereby the semiphosphorylated dimers adopted a conformation incapable of importin-α binding. This, in turn, substantially attenuated cardinal IFN-γ responses, including MHC expression, senescence, and antiparasitic immunity, and shifted the transcriptional output of IL-27 from STAT1 to STAT3. Our results uncover STAT2 as a pervasive cytokine regulator due to its inhibition of STAT1 in multiple signaling pathways and provide an understanding of the type 1 interferon-independent activities of this protein. [ABSTRACT FROM AUTHOR]

In the last 15 years, antiretroviral therapy (ART) has been the most globally impactful life-saving development of medical research. Antiretrovirals (ARVs) are used with great success for both the treatment and prevention of HIV infection. Despite these remarkable advances, this epidemic grows relentlessly worldwide. Over 2.1 million new infections occur each year, two-thirds in women and 240,000 in children. The widespread elimination of HIV will require the development of new, more potent prevention tools. Such efforts are imperative on a global scale. However, it must also be recognised that true containment of the epidemic requires the development and widespread implementation of a scientific advancement that has eluded us to date—a highly effective vaccine. Striving for such medical advances is what is required to achieve the end of AIDS. [ABSTRACT FROM AUTHOR]