Mild/asymptomatic COVID-19 in unvaccinated pregnant mothers impairs neonatal immune responses.

Maternal SARS-CoV-2 infection triggers placental inflammation and alters cord blood immune cell composition. However, most studies focus on outcomes of severe maternal infection. Therefore, we analyzed cord blood and chorionic villi from newborns of unvaccinated mothers who experienced mild/asymptomatic SARS-CoV-2 infection during pregnancy. We investigated immune cell rewiring using flow cytometry, single-cell RNA sequencing, and functional readouts using ex vivo stimulation with TLR agonists and pathogens. Maternal infection was associated with increased frequency of memory T and B cells and nonclassical monocytes in cord blood. Ex vivo T and B cell responses to stimulation were attenuated, suggesting a tolerogenic state. Maladaptive responses were also observed in cord blood monocytes, where antiviral responses were dampened but responses to bacterial TLRs were increased. Maternal infection was also associated with expansion and activation of placental Hofbauer cells, secreting elevated levels of myeloid cell-recruiting chemokines. Moreover, we reported increased activation of maternally derived monocytes/macrophages in the fetal placenta that were transcriptionally primed for antiviral responses. Our data indicate that even in the absence of vertical transmission or symptoms in the neonate, mild/asymptomatic maternal COVID-19 altered the transcriptional and functional state in fetal immune cells in circulation and in the placenta.

Multimodal profiling of term human decidua demonstrates immune adaptations with pregravid obesity.

Leukocyte diversity of the first-trimester maternal-fetal interface has been extensively described; however, the immunological landscape of the term decidua remains poorly understood. We therefore profiled human leukocytes from term decidua collected via scheduled cesarean delivery. Relative to the first trimester, our analyses show a shift from NK cells and macrophages to T cells and enhanced immune activation. Although circulating and decidual T cells are phenotypically distinct, they demonstrate significant clonotype sharing. We also report significant diversity within decidual macrophages, the frequency of which positively correlates with pregravid maternal body mass index. Interestingly, the ability of decidual macrophages to respond to bacterial ligands is reduced with pregravid obesity, suggestive of skewing toward immunoregulation as a possible mechanism to safeguard the fetus against excessive maternal inflammation. These findings are a resource for future studies investigating pathological conditions that compromise fetal health and reproductive success.

DNAJB6 isoform specific knockdown: Therapeutic potential for limb girdle muscular dystrophy D1.

Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved in vitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as in vivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional in vivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.

Mild/Asymptomatic Maternal SARS-CoV-2 Infection Leads to Immune Paralysis in Fetal Circulation and Immune Dysregulation in Fetal-Placental Tissues.

Few studies have addressed the impact of maternal mild/asymptomatic SARS-CoV-2 infection on the developing neonatal immune system. In this study, we analyzed umbilical cord blood and placental chorionic villi from newborns of unvaccinated mothers with mild/asymptomatic SARSCoV-2 infection during pregnancy using flow cytometry, single-cell transcriptomics, and functional assays. Despite the lack of vertical transmission, levels of inflammatory mediators were altered in cord blood. Maternal infection was also associated with increased memory T, B cells, and non-classical monocytes as well as increased activation. However, ex vivo responses to stimulation were attenuated. Finally, within the placental villi, we report an expansion of fetal Hofbauer cells and infiltrating maternal macrophages and rewiring towards a heightened inflammatory state. In contrast to cord blood monocytes, placental myeloid cells were primed for heightened antiviral responses. Taken together, this study highlights dysregulated fetal immune cell responses in response to mild maternal SARS-CoV-2 infection during pregnancy.

Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

SARS-CoV-2 Vaccine Booster Elicits Robust Prolonged Maternal Antibody Responses and Passive Transfer Via The Placenta And Breastmilk.

Background: Infection during pregnancy can result in adverse outcomes for both pregnant persons and offspring. Maternal vaccination is an effective mechanism to protect both mother and neonate into post-partum. However, our understanding of passive transfer of antibodies elicited by maternal SARS-CoV-2 mRNA vaccination during pregnancy remains incomplete. Objective: We aimed to evaluate the antibody responses engendered by maternal SARS-CoV-2 vaccination following initial and booster doses in maternal circulation and breastmilk to better understand passive immunization of the newborn. Study Design: We collected longitudinal blood samples from 121 pregnant women who received SARS-CoV-2 mRNA vaccines spanning from early gestation to delivery followed by collection of blood samples and breastmilk between delivery and 12 months post-partum. During the study, 70% of the participants also received a booster post-partum. Paired maternal plasma, breastmilk, umbilical cord plasma, and newborn plasma samples were tested via enzyme-linked immunosorbent assays (ELISA) to evaluate SARS-CoV-2 specific IgG antibody levels. Results: Vaccine-elicited maternal antibodies were detected in both cord blood and newborn blood, albeit at lower levels than maternal circulation, demonstrating transplacental passive immunization. Booster vaccination significantly increased spike specific IgG antibody titers in maternal plasma and breastmilk. Finally, SARS-CoV-2 specific IgG antibodies in newborn blood correlated negatively with days post initial maternal vaccine dose. Conclusion: Vaccine-induced maternal SARS-CoV-2 antibodies were passively transferred to the offspring in utero via the placenta and after birth via breastfeeding. Maternal booster vaccination, regardless of gestational age at maternal vaccination, significantly increased antibody levels in breastmilk and maternal plasma, indicating the importance of this additional dose to maximize passive protection against SARS-CoV-2 infection for neonates and infants until vaccination eligibility.

Disease-associated mutations within the yeast DNAJB6 homolog Sis1 slow conformer-specific substrate processing and can be corrected by the modulation of nucleotide exchange factors.

Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. Dominant mutations within DNAJB6 (Hsp40)-an Hsp70 co-chaperone-lead to a protein aggregation-linked myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights into the molecular basis of LGMDD1. Here, we show how mutations analogous to those found in LGMDD1 affect Sis1 (a functional homolog of human DNAJB6) function by altering the structure of client protein aggregates, interfering with the Hsp70 ATPase cycle, dimerization and substrate processing; poisoning the function of wild-type protein. These results uncover the mechanisms through which LGMDD1-associated mutations alter chaperone activity, and provide insights relevant to potential therapeutic interventions.

Monocytes and macrophages in pregnancy: The good, the bad, and the ugly.

A successful human pregnancy requires precisely timed adaptations by the maternal immune system to support fetal growth while simultaneously protecting mother and fetus against microbial challenges. The first trimester of pregnancy is characterized by a robust increase in innate immune activity that promotes successful implantation of the blastocyst and placental development. Moreover, early pregnancy is also a state of increased vulnerability to vertically transmitted pathogens notably, human immunodeficiency virus (HIV), Zika virus (ZIKV), SARS-CoV-2, and Listeria monocytogenes. As gestation progresses, the second trimester is marked by the establishment of an immunosuppressive environment that promotes fetal tolerance and growth while preventing preterm birth, spontaneous abortion, and other gestational complications. Finally, the period leading up to labor and parturition is characterized by the reinstatement of an inflammatory milieu triggering childbirth. These dynamic waves of carefully orchestrated changes have been dubbed the "immune clock of pregnancy." Monocytes in maternal circulation and tissue-resident macrophages at the maternal-fetal interface play a critical role in this delicate balance. This review will summarize the current data describing the longitudinal changes in the phenotype and function of monocyte and macrophage populations in healthy and complicated pregnancies.

Client processing is altered by novel myopathy-causing mutations in the HSP40 J domain.

The misfolding and aggregation of proteins is often implicated in the development and progression of degenerative diseases. Heat shock proteins (HSPs), such as the ubiquitously expressed Type II Hsp40 molecular chaperone, DNAJB6, assist in protein folding and disaggregation. Historically, mutations within the DNAJB6 G/F domain have been associated with Limb-Girdle Muscular Dystrophy type 1D, now referred to as LGMDD1, a dominantly inherited degenerative disease. Recently, novel mutations within the J domain of DNAJB6 have been reported in patients with LGMDD1. Since novel myopathy-causing mutations in the Hsp40 J domain have yet to be characterized and both the function of DNAJB6 in skeletal muscle and the clients of this chaperone are unknown, we set out to assess the effect of these mutations on chaperone function using the genetically tractable yeast system. The essential yeast Type II Hsp40, Sis1, is homologous to DNAJB6 and is involved in the propagation of yeast prions. Using phenotypic, biochemical, and functional assays we found that homologous mutations in the Sis1 J domain differentially alter the processing of specific yeast prion strains, as well as a non-prion substrate. These data suggest that the newly-identified mutations in the J domain of DNAJB6 cause aberrant chaperone function that leads to the pathogenesis in LGMDD1.

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy.

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.

Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D.

OBJECTIVE: To understand DNAJB6's function in skeletal muscle and identify therapeutic targets for limb-girdle muscular dystrophy 1D (LGMD1D). METHODS: DNAJB6 knockout (KO) myoblasts were generated with Crispr/cas9 technology, and differentially accumulated proteins were identified using stable isotope labeling, followed by quantitative mass spectrometry. Cultured KO myotubes and mouse muscle from DNAJB6b-WT or DNAJB6b-F93L mice were analyzed using histochemistry, immunohistochemistry, and immunoblot. Mouse functional strength measures included forelimb grip strength and inverted wire hang. RESULTS: DNAJB6 inactivation leads to the accumulation of sarcomeric proteins and hypertrophic myotubes with an enhanced fusion index. The increased fusion in DNAJB6 KO myotubes correlates with diminished glycogen synthase kinase-ß (GSK3ß) activity. In contrast, LGMD1D mutations in DNAJB6 enhance GSK3ß activation and suppress ß-catenin and NFAT3c signaling. GSK3ß inhibition with lithium chloride improves muscle size and strength in an LGMD1D preclinical mouse model. CONCLUSIONS: Our results suggest that DNAJB6 facilitates protein quality control and negatively regulates myogenic signaling. In addition, LGMD1D-associated DNAJB6 mutations inhibit myogenic signaling through augmented GSK3ß activity. GSK3ß inhibition with lithium chloride may be a therapeutic option in LGMD1D.

A toxic imbalance of Hsp70s in Saccharomyces cerevisiae is caused by competition for cofactors.

Molecular chaperones are responsible for managing protein folding from translation through degradation. These crucial machines ensure that protein homeostasis is optimally maintained for cell health. However, 'too much of a good thing' can be deadly, and the excess of chaperones can be toxic under certain cellular conditions. For example, overexpression of Ssa1, a yeast Hsp70, is toxic to cells in folding-challenged states such as [PSI+]. We discovered that overexpression of the nucleotide exchange factor Sse1 can partially alleviate this toxicity. We further argue that the basis of the toxicity is related to the availability of Hsp70 cofactors, such as Hsp40 J-proteins and nucleotide exchange factors. Ultimately, our work informs future studies about functional chaperone balance and cautions against therapeutic chaperone modifications without a thorough examination of cofactor relationships.

Heterologous prion-forming proteins interact to cross-seed aggregation in Saccharomyces cerevisiae.

The early stages of protein misfolding remain incompletely understood, as most mammalian proteinopathies are only detected after irreversible protein aggregates have formed. Cross-seeding, where one aggregated protein templates the misfolding of a heterologous protein, is one mechanism proposed to stimulate protein aggregation and facilitate disease pathogenesis. Here, we demonstrate the existence of cross-seeding as a crucial step in the formation of the yeast prion [PSI +], formed by the translation termination factor Sup35. We provide evidence for the genetic and physical interaction of the prion protein Rnq1 with Sup35 as a predominant mechanism leading to self-propagating Sup35 aggregation. We identify interacting sites within Rnq1 and Sup35 and determine the effects of breaking and restoring a crucial interaction. Altogether, our results demonstrate that single-residue disruption can drastically reduce the effects of cross-seeding, a finding that has important implications for human protein misfolding disorders.

Rapid generation of hypomorphic mutations.

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones.

The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders.

Amyloid Prions in Fungi.

Prions are infectious protein polymers that have been found to cause fatal diseases in mammals. Prions have also been identified in fungi (yeast and filamentous fungi), where they behave as cytoplasmic non-Mendelian genetic elements. Fungal prions correspond in most cases to fibrillary ß-sheet-rich protein aggregates termed amyloids. Fungal prion models and, in particular, yeast prions were instrumental in the description of fundamental aspects of prion structure and propagation. These models established the "protein-only" nature of prions, the physical basis of strain variation, and the role of a variety of chaperones in prion propagation and amyloid aggregate handling. Yeast and fungal prions do not necessarily correspond to harmful entities but can have adaptive roles in these organisms.

Myofibrillar disruption and RNA-binding protein aggregation in a mouse model of limb-girdle muscular dystrophy 1D.

Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominantly inherited missense mutations in DNAJB6, an Hsp40 co-chaperone. LGMD1D muscle has rimmed vacuoles and inclusion bodies containing DNAJB6, Z-disc proteins and TDP-43. DNAJB6 is expressed as two isoforms; DNAJB6a and DNAJB6b. Both isoforms contain LGMD1D mutant residues and are expressed in human muscle. To identify which mutant isoform confers disease pathogenesis and generate a mouse model of LGMD1D, we evaluated DNAJB6 expression and localization in skeletal muscle as well as generating DNAJB6 isoform specific expressing transgenic mice. DNAJB6a localized to myonuclei while DNAJB6b was sarcoplasmic. LGMD1D mutations in DNAJB6a or DNAJB6b did not alter this localization in mouse muscle. Transgenic mice expressing the LGMD1D mutant, F93L, in DNAJB6b under a muscle-specific promoter became weak, had early lethality and developed muscle pathology consistent with myopathy after 2 months; whereas mice expressing the same F93L mutation in DNAJB6a or overexpressing DNAJB6a or DNAJB6b wild-type transgenes remained unaffected after 1 year. DNAJB6b localized to the Z-disc and DNAJB6b-F93L expressing mouse muscle had myofibrillar disorganization and desmin inclusions. Consistent with DNAJB6 dysfunction, keratin 8/18, a DNAJB6 client also accumulated in DNAJB6b-F93L expressing mouse muscle. The RNA-binding proteins hnRNPA1 and hnRNPA2/B1 accumulated and co-localized with DNAJB6 at sarcoplasmic stress granules suggesting that these proteins maybe novel DNAJB6b clients. Similarly, hnRNPA1 and hnRNPA2/B1 formed sarcoplasmic aggregates in patients with LGMD1D. Our data support that LGMD1D mutations in DNAJB6 disrupt its sarcoplasmic function suggesting a role for DNAJB6b in Z-disc organization and stress granule kinetics.