The NLRP3 inflammasome is essential for caspase-1 activation and the release of interleukin (IL)-1β, IL-18, and gasdermin-D in myeloid cells. However, research on species-specific NLRP3's physiological impact is limited. We engineer mice with the human NLRP3 gene, driven by either the human or mouse promoter, via syntenic replacement at the mouse Nlrp3 locus. Both promoters facilitate hNLRP3 expression in myeloid cells, but the mouse promoter responds more robustly to LPS. Investigating the disease impact of differential NLRP3 regulation, we introduce the D305N gain-of-function mutation into both humanized lines. Chronic inflammation is evident with both promoters; however, CNS outcomes vary significantly. Despite poor response to LPS, the human promoter results in D305N-associated aseptic meningitis, mirroring human pathology. The mouse promoter, although leading to increased CNS expression post-LPS, does not induce meningitis in D305N mutants. Therefore, human-like NLRP3 expression may be crucial for accurate modeling of its role in disease pathogenesis.

CD4+ Foxp3+ T regulatory (Treg) cells are key players in preventing lethal autoimmunity. Tregs undertake differentiation processes and acquire diverse functional properties. However, how Treg's differentiation and functional specification are regulated remains incompletely understood. Here, we report that gradient expression of TCF1 and LEF1 distinguishes Tregs into three distinct subpopulations, particularly highlighting a subset of activated Treg (aTreg) cells. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to systemic autoimmunity. TCF1 and LEF1 are dispensable for Treg's suppressive capacity but essential for maintaining a normal aTreg pool and promoting Treg's competitive survival. As a consequence, the development of T follicular regulatory (Tfr) cells, which are a subset of aTreg, is abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmunity.

Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a-/- and Epm2b-/-) and one of APBD (Gbe1ys/ys), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.

Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

Virus-specific humoral and cellular immunity act synergistically to protect the host from viral infection. We interrogate the dynamic changes of virological and immunological parameters in 12 patients with symptomatic acute SARS-CoV-2 infection from disease onset to convalescence or death. We quantify SARS-CoV-2 viral RNA in the respiratory tract in parallel with antibodies and circulating T cells specific for various structural (nucleoprotein [NP], membrane [M], ORF3a, and spike) and non-structural (ORF7/8, NSP7, and NSP13) proteins. Although rapid induction and quantity of humoral responses associate with an increase in disease severity, early induction of interferon (IFN)-γ-secreting SARS-CoV-2-specific T cells is present in patients with mild disease and accelerated viral clearance. These findings provide support for the prognostic value of early functional SARS-CoV-2-specific T cells with important implications in vaccine design and immune monitoring.

Zika virus (ZIKV) is an Aedes-mosquito-borne flavivirus that causes debilitating congenital and developmental disorders. Improved understanding of ZIKV pathogenesis could assist efforts to fill the therapeutic and vaccine gap. We use several ZIKV strains, including a pair differing by a single phenylalanine-to-leucine substitution (M-F37L) in the membrane (M) protein, coupled with unbiased genomics to demarcate the border between attenuated and pathogenic infection. We identify infection-induced metabolic dysregulation as a minimal set of host alterations that differentiates attenuated from pathogenic ZIKV strains. Glycolytic rewiring results in impaired oxidative phosphorylation and mitochondrial dysfunction that trigger inflammation and apoptosis in pathogenic but not attenuated ZIKV strains. Critically, pyruvate supplementation prevents cell death, in vitro, and rescues fetal development in ZIKV-infected dams. Our findings thus demonstrate dysregulated metabolism as an underpinning of ZIKV pathogenicity and raise the potential of pyruvate supplementation in expectant women as a prophylaxis against congenital Zika syndrome.

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.

LACC1 genetic variants are associated with multiple immune-mediated diseases. However, laccase domain containing-1 (LACC1) functions are incompletely defined. We find that upon stimulation of the pattern-recognition receptor (PRR) NOD2, LACC1 localizes to the endoplasmic reticulum (ER) and forms a complex with ER-stress sensors. All three ER-stress branches, PERK, IRE1α, and ATF6, are required for NOD2-induced signaling, cytokines, and antimicrobial pathways in human macrophages. LACC1, and its localization to the ER, is required for these outcomes. Relative to wild-type (WT) LACC1, transfection of the common Val254 and rare Arg284 immune-mediated disease-risk LACC1 variants into HeLa cells and macrophages, as well as macrophages from LACC1 Val254 carriers, shows reduced NOD2-induced ER stress-associated outcomes; these downstream outcomes are restored by rescuing ER stress. Therefore, we identify ER stress to be essential in PRR-induced outcomes in macrophages, define a critical role for LACC1 in these ER stress-dependent events, and elucidate how LACC1 disease-risk variants mediate these outcomes.

Megakaryocytes (MKs) come from a complex process of hematopoietic progenitor maturation within the bone marrow that gives rise to de novo circulating platelets. Bone marrow microenvironment contains a large number of adipocytes with a still ill-defined role. This study aims to analyze the influence of adipocytes and increased medullar adiposity in megakaryopoiesis. An in vivo increased medullar adiposity in mice caused by high-fat-diet-induced obesity is associated to an enhanced MK maturation and proplatelet formation. In vitro co-culture of adipocytes with bone marrow hematopoietic progenitors shows that delipidation of adipocytes directly supports MK maturation by enhancing polyploidization, amplifying the demarcation membrane system, and accelerating proplatelet formation. This direct crosstalk between adipocytes and MKs occurs through adipocyte fatty acid transfer to MKs involving CD36 to reinforce megakaryocytic maturation. Thus, these findings unveil an influence of adiposity on MK homeostasis based on a dialogue between adipocytes and MKs.

FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer.

Bone stromal cells are critical for bone homeostasis and regeneration. Growing evidence suggests that non-stem bone niche cells support bone homeostasis and regeneration via paracrine mechanisms, which remain to be elucidated. Here, we show that physiologically quiescent SM22α-lineage stromal cells expand after bone injury to regulate diverse processes of intramembranous bone regeneration. The majority of SM22α-lineage cells neither act as stem cells in vivo nor show their expression patterns. Dysfunction of SM22α-lineage niche cells induced by loss of platelet-derived growth factor receptor β (PDGFRβ) impairs bone repair. We further show that PDGFRβ-triggered hydrogen sulfide (H2S) generation in SM22α-lineage niche cells facilitates osteogenesis and angiogenesis and suppresses overactive osteoclastogenesis. Collectively, these data demonstrate that non-stem SM22α-lineage niche cells support the niche for bone regeneration with a PDGFRβ/H2S-dependent regulatory mechanism. Our findings provide further insight into non-stem bone stromal niche cell populations and niche-regulation strategy for bone repair.

Human and mouse thermal physiology differ due to dissimilar body sizes. Unexpectedly, in mice we found no ambient temperature zone where both metabolic rate and body temperature were constant. Body temperature began increasing once cold-induced thermogenesis was no longer required. This result reproduced in male, female, C57BL/6J, 129, chow-fed, diet-induced obese, and ob/ob mice as well as Trpv1-/-;Trpm8-/-;Trpa1-/- mice lacking thermal sensory channels. During the resting-light phase, the energy expenditure minimum spanned ∼4°C of ambient temperature, whereas in the active-dark phase it approximated a point. We propose the concept of a thermoneutral point (TNP), a discrete ambient temperature below which energy expenditure increases and above which body temperature increases. Humans do not have a TNP. As studied, the mouse TNP is ∼29°C in light phase and ∼33°C in dark phase. These observations inform how thermoneutrality is defined and how mice are used to model human energy physiology and drug development.

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targeted approach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantage of DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial, to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed mutagenesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) protein that reduces 16681 infection and dissemination in both Aedes aegypti, as well as in mammalian cells to produce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a known host factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 and thus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights on DENV attenuation on a clinically tested strain.

Multidonor antibodies are of interest for vaccine design because they can in principle be elicited in the general population by a common set of immunogens. For influenza, multidonor antibodies have been observed against the hemagglutinin (HA) stem, but not the immunodominant HA head. Here, we identify and characterize a multidonor antibody class (LPAF-a class) targeting the HA head. This class exhibits potent viral entry inhibition against H1N1 A/California/04/2009 (CA09) virus. LPAF-a class antibodies derive from the HV2-70 gene and contain a "Tyr-Gly-Asp"-motif, which occludes the HA-sialic acid binding site as revealed by a co-crystal structure with HA. Both germline-reverted and mature LPAF antibodies potently neutralize CA09 virus and have nanomolar affinities for CA09 HA. Moreover, increased frequencies for LPFA-a class antibodies are observed in humans after a single vaccination. Overall, this work highlights the identification of a multidonor class of head-directed influenza-neutralizing antibodies and delineates the mechanism of their recurrent elicitation in humans.

Although the antibody response to COVID-19 vaccination has been studied extensively at the polyclonal level using immune sera, little has been reported on the antibody response at the monoclonal level. Here, we isolate a panel of 44 anti-SARS-CoV-2 monoclonal antibodies (mAbs) from an individual who received two doses of the ChAdOx1 nCoV-19 (AZD1222) vaccine at a 12-week interval. We show that, despite a relatively low serum neutralization titer, Spike-reactive IgG+ B cells are still detectable 9 months post-boost. Furthermore, mAbs with potent neutralizing activity against the current SARS-CoV-2 variants of concern (Alpha, Gamma, Beta, Delta, and Omicron) are present. The vaccine-elicited neutralizing mAbs form eight distinct competition groups and bind epitopes overlapping with neutralizing mAbs elicited following SARS-CoV-2 infection. AZD1222-elicited mAbs are more mutated than mAbs isolated from convalescent donors 1-2 months post-infection. These findings provide molecular insights into the AZD1222 vaccine-elicited antibody response.

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2∗02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

Dengue virus (DENV) is a human arboviral pathogen accounting for 390 million infections every year. The available vaccine has limited efficacy, and DENV-specific drugs have not been generated. To better understand DENV-host cell interaction, we employed RNA interference-based screening of the human kinome and identified fibroblast growth factor receptor 4 (FGFR4) to control the DENV replication cycle. Pharmacological inhibition of FGFR exerts a reciprocal effect by reducing DENV RNA replication and promoting the production of infectious virus particles. Addressing the latter effect, we found that the FGFR signaling pathway modulates intracellular distribution of DENV particles in a PI3K-dependent manner. Upon FGFR inhibition, virions accumulate in the trans-Golgi network compartment, where they undergo enhanced maturation cleavage of the envelope protein precursor membrane (prM), rendering virus particles more infectious. This study reveals an unexpected reciprocal role of a cellular receptor tyrosine kinase regulating DENV RNA replication and the production of infectious virions.