bims-nimamd Biomed News
on Neuroimmunity and neuroinflammation in ageing and metabolic disease
Issue of 2022‒02‒20
sixty-four papers selected by
Fawaz Alzaïd
Sorbonne Université

  1. J Immunol. 2022 Feb 14. pii: ji2100741. [Epub ahead of print]
      Alveolar macrophages (AMs) are major lung tissue-resident macrophages capable of proliferating and self-renewal in situ. AMs are vital in pulmonary antimicrobial immunity and surfactant clearance. The mechanisms regulating AM compartment formation and maintenance remain to be fully elucidated currently. In this study, we have explored the roles of mitochondrial transcription factor A (TFAM)-mediated mitochondrial fitness and metabolism in regulating AM formation and function. We found that TFAM deficiency in mice resulted in significantly reduced AM numbers and impaired AM maturation in vivo. TFAM deficiency was not required for the generation of AM precursors nor the differentiation of AM precursors into AMs, but was critical for the maintenance of AM compartment. Mechanistically, TFAM deficiency diminished gene programs associated with AM proliferation and self-renewal and promoted the expression of inflammatory genes in AMs. We further showed that TFAM-mediated AM compartment impairment resulted in defective clearance of cellular debris and surfactant in the lung and increased the host susceptibility to severe influenza virus infection. Finally, we found that influenza virus infection in AMs led to impaired TFAM expression and diminished mitochondrial fitness and metabolism. Thus, our data have established the critical function of TFAM-mediated mitochondrial metabolism in AM maintenance and function.
  2. JCI Insight. 2022 Feb 15. pii: e157694. [Epub ahead of print]
      Altered islet architecture is associated with β cell dysfunction and Type 2 Diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed "re-activated" β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in post-natal β cells impaired glucose stimulated insulin secretion (GSIS) and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion, and impaired murine and human pseudo-islet formation. Consistent with our mouse data, Notch and Ephrin signaling are increased in metabolically-inflexible β cells in patients with T2D. These studies suggest than islet architecture can be permanently altered by β cell Notch/Ephrin signaling during a morphogenetic window in early life.
    Keywords:  Cell migration/adhesion; Diabetes; Endocrinology; Islet cells
  3. Hepatology. 2022 Feb 17.
      The concept of hepatocyte functional zonation is well established, with differences in metabolism and xenobiotic processing determined by multiple factors including oxygen and nutrient levels across the hepatic lobule. However, recent advances in single cell genomics technologies, including single cell and nuclei RNA sequencing, and the rapidly evolving fields of spatial transcriptomic and proteomic profiling have greatly increased our understanding of liver zonation. Here we discuss how these transformative experimental strategies are being leveraged to dissect liver zonation at unprecedented resolution, and how this new information should facilitate the emergence of novel precision medicine-based therapies for patients with liver disease.
  4. Nat Immunol. 2022 Feb 17.
      T cells acquire a regulatory phenotype when their T cell antigen receptors (TCRs) experience an intermediate- to high-affinity interaction with a self-peptide presented via the major histocompatibility complex (MHC). Using TCRβ sequences from flow-sorted human cells, we identified TCR features that promote regulatory T cell (Treg) fate. From these results, we developed a scoring system to quantify TCR-intrinsic regulatory potential (TiRP). When applied to the tumor microenvironment, TiRP scoring helped to explain why only some T cell clones maintained the conventional T cell (Tconv) phenotype through expansion. To elucidate drivers of these predictive TCR features, we then examined the two elements of the Treg TCR ligand separately: the self-peptide and the human MHC class II molecule. These analyses revealed that hydrophobicity in the third complementarity-determining region (CDR3β) of the TCR promotes reactivity to self-peptides, while TCR variable gene (TRBV gene) usage shapes the TCR's general propensity for human MHC class II-restricted activation.
  5. Nat Commun. 2022 Feb 16. 13(1): 888
      Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.
  6. Immunol Rev. 2022 Feb 17.
      Random VDJ recombination early in T and B cell development enables the adaptive immune system to recognize a vast array of evolving pathogens via antigen receptors. However, the potential of such randomly generated TCRs and BCRs to recognize and respond to self-antigens requires layers of tolerance mechanisms to mitigate the risk of life-threatening autoimmunity. Since they were originally cloned more than three decades ago, the NR4A family of nuclear hormone receptors have been implicated in many critical aspects of immune tolerance, including negative selection of thymocytes, peripheral T cell tolerance, regulatory T cells (Treg), and most recently in peripheral B cell tolerance. In this review, we discuss important insights from many laboratories as well as our own group into the function and mechanisms by which this small class of primary response genes promotes self-tolerance and immune homeostasis to balance the need for host defense against the inherent risks posed by the adaptive immune system.
    Keywords:   Nr4a1 ; Nr4a3 ; B cell tolerance; NOR1; NUR77; T cell tolerance; anergy; clonal diversity; immunodominance; negative selection; self-reactivity
  7. Nat Cell Biol. 2022 Feb;24(2): 135-147
      Ageing organisms accumulate senescent cells that are thought to contribute to body dysfunction. Telomere shortening and damage are recognized causes of cellular senescence and ageing. Several human conditions associated with normal ageing are precipitated by accelerated telomere dysfunction. Here, we systematize a large body of evidence and propose a coherent perspective to recognize the broad contribution of telomeric dysfunction to human pathologies.
  8. FASEB J. 2022 Mar;36(3): e22200
      Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2 mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2 mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.
    Keywords:  Crohn's disease; Paneth cell; alpha-defensin; innate immunity; intelectin; lectin; metaplasia; omentin; small intestine; ulcerative colitis
  9. J Immunol. 2022 Feb 18. pii: ji2100414. [Epub ahead of print]
      E-protein transcription factors limit group 2 innate lymphoid cell (ILC2) development while promoting T cell differentiation from common lymphoid progenitors. Inhibitors of DNA binding (ID) proteins block E-protein DNA binding in common lymphoid progenitors to allow ILC2 development. However, whether E-proteins influence ILC2 function upon maturity and activation remains unclear. Mice that overexpress ID1 under control of the thymus-restricted proximal Lck promoter (ID1tg/WT) have a large pool of primarily thymus-derived ILC2s in the periphery that develop in the absence of E-protein activity. We used these mice to investigate how the absence of E-protein activity affects ILC2 function and the genomic landscape in response to house dust mite (HDM) allergens. ID1tg/WT mice had increased KLRG1- ILC2s in the lung compared with wild-type (WT; ID1WT/WT) mice in response to HDM, but ID1tg/WT ILC2s had an impaired capacity to produce type 2 cytokines. Analysis of WT ILC2 accessible chromatin suggested that AP-1 and C/EBP transcription factors but not E-proteins were associated with ILC2 inflammatory gene programs. Instead, E-protein binding sites were enriched at functional genes in ILC2s during development that were later dynamically regulated in allergic lung inflammation, including genes that control ILC2 response to cytokines and interactions with T cells. Finally, ILC2s from ID1tg/WT compared with WT mice had fewer regions of open chromatin near functional genes that were enriched for AP-1 factor binding sites following HDM treatment. These data show that E-proteins shape the chromatin landscape during ILC2 development to dictate the functional capacity of mature ILC2s during allergic inflammation in the lung.
  10. J Hepatol. 2022 Feb 12. pii: S0168-8278(22)00077-0. [Epub ahead of print]
      BACKGROUND & AIMS: The pathogenesis of liver fibrosis requires activation of hepatic stellate cells (HSCs); once activated, HSCs lose intracellular fatty acids but the role of fatty acid oxidation and carnitine palmitoyltransferase 1A (CPT1A) in this process remains largely unexplored.METHODS: CPT1A was found in HSCs of patients with fibrosis. Pharmacological and genetic manipulation of CPT1A were done in HSCs human cell lines and primary HCSs. Finally, we induced fibrosis in mice lacking CPT1A specifically in HSCs.
    RESULTS: Here we show that CPT1A expression is elevated in HSCs of patients with NASH, showing a positive correlation with the fibrosis score. This was corroborated in rodents with fibrosis, as well as in primary human HSCs and LX-2 cells activated by transforming growth factor β1 (TGFβ1) and fetal bovine serum (FBS). Furthermore, both pharmacological and genetic silencing of CPT1A prevent TGFβ1- and FBS-induced HSC activation by reducing mitochondrial activity. The overexpression of CPT1A, induced by saturated fatty acids and reactive oxygen species, triggers mitochondrial function and the expression of fibrogenic markers. Finally, mice lacking CPT1A specifically in HSCs are protected against fibrosis, induced by a choline-deficient high fat diet, a methionine- and choline-deficient diet, or treatment with carbon tetrachloride.
    CONCLUSIONS: These results indicate that CPT1A plays a critical role in activation of HSCs and is implicated in the development of liver fibrosis, making it a potentially actionable target for fibrosis treatment.
    LAY SUMMARY: We show that CPT1A located in HSCs is elevated in patients and mice models with fibrosis, and that CPT1A induces the activation of these cells. Inhibition of CPT1A ameliorates fibrosis by preventing the activation of HSCs.
    Keywords:  CPT1A; NASH; beta oxidation; fatty acids; fibrosis; metabolism
  11. Endocrinology. 2022 Feb 16. pii: bqac018. [Epub ahead of print]
      Mitochondrial dysfunction in adipose tissue has been associated with type 2 diabetes, but it is unclear whether it is a cause or the consequence. Mitochondrial complex I is a major site of reactive oxygen species generation and a therapeutic target. Here we report that genetic deletion of the complex I subunit Ndufs4 specifically in adipose tissue results in an increased propensity to develop diet-induced weight gain, glucose intolerance, and elevated levels of fat inflammatory genes. This outcome is apparent in young males but not in young females, suggesting that females are relatively protected from the adverse consequences of adipose mitochondrial dysfunction for metabolic health. Mutant mice of both sexes exhibit defects in brown adipose tissue thermogenesis. Fibroblast growth factor 21 (FGF21) signaling in adipose tissue is selectively blunted in male mutant mice relative to wild-type littermates, consistent with sex-dependent regulation of its autocrine/paracrine action in adipocytes. Together, these findings support that adipocyte-specific mitochondrial dysfunction is sufficient to induce tissue inflammation and can cause systemic glucose abnormalities in male mice.
    Keywords:  FGF21; Ndufs4; impaired glucose tolerance; inflammation; mitochondria
  12. J Clin Invest. 2022 Feb 17. pii: e151725. [Epub ahead of print]
      The gut microbiome shapes local and systemic immunity. The liver is presumed to be a protected sterile site. As such, a hepatic microbiome has not been examined. Here, we showed a liver microbiome in mice and humans that is distinct from the gut and is enriched in Proteobacteria. It undergoes dynamic alterations with age and is influenced by the environment and host physiology. Fecal microbial transfer experiments revealed that the liver microbiome is populated from the gut in a highly selective manner. Hepatic immunity is dependent on the microbiome, specifically Bacteroidetes species. Targeting Bacteroidetes with oral antibiotics reduced hepatic immune cells by ~90%, prevented APC maturation, and mitigated adaptive immunity. Mechanistically, our findings are consistent with presentation of Bacteroidetes-derived glycosphingolipids to NKT cells promoting CCL5 signaling, which drives hepatic leukocyte expansion and activation, among other possible host-microbe interactions. Collectively, we reveal a microbial - glycosphingolipid - NKT - CCL5 axis that underlies hepatic immunity.
    Keywords:  Antigen; Chemokines; Hepatology; Innate immunity; Microbiology
  13. J Clin Invest. 2022 02 15. pii: e152859. [Epub ahead of print]132(4):
      Patients with heart failure (HF) have augmented vascular tone, which increases cardiac workload, impairing ventricular output and promoting further myocardial dysfunction. The molecular mechanisms underlying the maladaptive vascular responses observed in HF are not fully understood. Vascular smooth muscle cells (VSMCs) control vasoconstriction via a Ca2+-dependent process, in which the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) on the sarcoplasmic reticulum (SR) plays a major role. To dissect the mechanistic contribution of intracellular Ca2+ release to the increased vascular tone observed in HF, we analyzed the remodeling of IP3R1 in aortic tissues from patients with HF and from controls. VSMC IP3R1 channels from patients with HF and HF mice were hyperphosphorylated by both serine and tyrosine kinases. VSMCs isolated from IP3R1VSMC-/- mice exhibited blunted Ca2+ responses to angiotensin II (ATII) and norepinephrine compared with control VSMCs. IP3R1VSMC-/- mice displayed significantly reduced responses to ATII, both in vivo and ex vivo. HF IP3R1VSMC-/- mice developed significantly less afterload compared with HF IP3R1fl/fl mice and exhibited significantly attenuated progression toward decompensated HF and reduced interstitial fibrosis. Ca2+-dependent phosphorylation of the MLC by MLCK activated VSMC contraction. MLC phosphorylation was markedly increased in VSMCs from patients with HF and HF mice but reduced in VSMCs from HF IP3R1VSMC-/- mice and HF WT mice treated with ML-7. Taken together, our data indicate that VSMC IP3R1 is a major effector of increased vascular tone, which contributes to increased cardiac afterload and decompensation in HF.
    Keywords:  Calcium channels; Calcium signaling; Cardiology; Cardiovascular disease; Cell Biology
  14. N Engl J Med. 2022 Feb 17. pii: 10.1056/NEJMc2114590#sa2. [Epub ahead of print]386(7): e17
  15. J Clin Invest. 2022 Mar 01. pii: e158251. [Epub ahead of print]132(5):
      Sickle cell disease (SCD) is associated with an increased risk of vascular-occlusive events and of leukemia. Clonal hematopoiesis (CH) may increase both risks. In turn, physiologic abnormalities in SCD may modify the incidence and/or distribution of genetic alterations in CH. In a recent issue of the JCI, Liggett et al. found no difference in CH rate between individuals with versus without SCD. Here we contextualize this report and discuss the complex interplay between CH and SCD with particular attention to consequences for emerging gene therapies. We further consider the limitations in our current understanding of these topics that must be addressed in order to optimize therapeutic strategies for SCD.
  16. J Immunol. 2022 Feb 14. pii: ji2100550. [Epub ahead of print]
      Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.
  17. Sci Rep. 2022 Feb 18. 12(1): 2834
      MicroRNAs (miRNAs) are non-coding small RNAs which play a critical role in the regulation of gene expression in cells. It is known that miRNAs are often expressed as multiple isoforms, called isomiRs, which may have alternative regulatory functions. Despite the recent development of several single cell small RNA sequencing protocols, these methods have not been leveraged to investigate isomiR expression and regulation to better understand their role on a single cell level. Here we integrate sequencing data from three independent studies and find substantial differences in isomiR composition that suggest that cell autonomous mechanisms may drive isomiR processing. We also find evidence of altered regulatory functions of different classes of isomiRs, when compared to their respective wild-type miRNA, which supports a biological role for many of the isomiRs that are expressed.
  18. Blood. 2022 Feb 14. pii: blood.2021014654. [Epub ahead of print]
      Both innate and adaptive lymphocytes have critical roles in mucosal defense that contain commensal microbial communities and protect against pathogen invasion. Here we characterize mucosal immunity in human severe combined immunodeficiency (SCID) patients receiving hematopoietic stem cell transplantation (HSCT) with or without myeloablation. We confirmed that pre-transplant conditioning impacted on innate (NK, ILC) and adaptive (B and T cells) lymphocyte reconstitution in these SCID patients and now demonstrate that this further extends to generation of Th2 and Tc2 cells. Using an integrated approach to assess nasopharyngeal immunity, we identify a local mucosal defect in type 2 cytokines, mucus production and a selective local IgA deficiency in HSCT-treated SCID patients with genetic defects in IL2RG/GC or JAK3. These patients have a reduction in IgA-coated nasopharyngeal bacteria and exhibit microbial dysbiosis with increased pathobiont carriage. Interestingly, IVIG replacement therapy can partially normalize nasopharyngeal Ig profiles and restore microbial communities in GC/JAK3 patients. Together, our results suggest a potential non-redundant role for type 2 immunity and/or of local IgA antibody production in the maintenance of nasopharyngeal microbial homeostasis and mucosal barrier function.
  19. Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2116588119. [Epub ahead of print]119(8):
    CMV Systems Immunobiology Group
      Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.
    Keywords:  NK cell; cytomegalovirus; cytotoxic lymphocyte; mass cytometry; renal transplantation
  20. Front Immunol. 2022 ;13 835994
      CCR5, a chemokine receptor central for orchestrating lymphocyte/cell migration to the sites of inflammation and to the immunosurveillance, is involved in the pathogenesis of a wide spectrum of health conditions, including inflammatory diseases, viral infections, cancers and autoimmune diseases. CCR5 is also the primary coreceptor for the human immunodeficiency viruses (HIVs), supporting its entry into CD4+ T lymphocytes upon transmission and in the early stages of infection in humans. A natural loss-of-function mutation CCR5-Δ32, preventing the mutated protein expression on the cell surface, renders homozygous carriers of the null allele resistant to HIV-1 infection. This phenomenon was leveraged in the development of therapies and cure strategies for AIDS. Meanwhile, over 40 African nonhuman primate species are long-term hosts of simian immunodeficiency virus (SIV), an ancestral family of viruses that give rise to the pandemic CCR5 (R5)-tropic HIV-1. Many natural hosts typically do not progress to immunodeficiency upon the SIV infection. They have developed various strategies to minimize the SIV-related pathogenesis and disease progression, including an array of mechanisms employing modulation of the CCR5 receptor activity: (i) deletion mutations abrogating the CCR5 surface expression and conferring resistance to infection in null homozygotes; (ii) downregulation of CCR5 expression on CD4+ T cells, particularly memory cells and cells at the mucosal sites, preventing SIV from infecting and killing cells important for the maintenance of immune homeostasis, (iii) delayed onset of CCR5 expression on the CD4+ T cells during ontogenetic development that protects the offspring from vertical transmission of the virus. These host adaptations, aimed at lowering the availability of target CCR5+ CD4+ T cells through CCR5 downregulation, were countered by SIV, which evolved to alter the entry coreceptor usage toward infecting different CD4+ T-cell subpopulations that support viral replication yet without disruption of host immune homeostasis. These natural strategies against SIV/HIV-1 infection, involving control of CCR5 function, inspired therapeutic approaches against HIV-1 disease, employing CCR5 coreceptor blocking as well as gene editing and silencing of CCR5. Given the pleiotropic role of CCR5 in health beyond immune disease, the precision as well as costs and benefits of such interventions needs to be carefully considered.
    Keywords:  African green monkey; CCR5; delta 32; human immunodeficiency virus; red-capped mangabey; simian immunodeficiency virus; sooty mangabey; virus transmission
  21. J Immunol. 2022 Feb 18. pii: ji2101041. [Epub ahead of print]
      Type III IFNs (IFNLs) are newly discovered cytokines, acting at epithelial and other barriers, that exert immunomodulatory functions in addition to their primary roles in antiviral defense. In this study, we define a role for IFNLs in maintaining autoreactive T cell effector function and limiting recovery in a murine model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis. Genetic or Ab-based neutralization of the IFNL receptor (IFNLR) resulted in lack of disease maintenance during experimental autoimmune encephalomyelitis, with loss of CNS Th1 effector responses and limited axonal injury. Phenotypic effects of IFNLR signaling were traced to increased APC function, with associated increase in T cell production of IFN-γ and GM-CSF. Consistent with this, IFNL levels within lesions of CNS tissues derived from patients with MS were elevated compared with MS normal-appearing white matter. Furthermore, expression of IFNLR was selectively elevated in MS active lesions compared with inactive lesions or normal-appearing white matter. These findings suggest IFNL signaling as a potential therapeutic target to prevent chronic autoimmune neuroinflammation.
  22. Cell Rep. 2022 02 15. pii: S2211-1247(22)00084-5. [Epub ahead of print]38(7): 110363
      Thymic atrophy reduces naive T cell production and contributes to increased susceptibility to viral infection with age. Expression of tissue-restricted antigen (TRA) genes also declines with age and has been thought to increase autoimmune disease susceptibility. We find that diminished expression of a model TRA gene in aged thymic stromal cells correlates with impaired clonal deletion of cognate T cells recognizing an autoantigen involved in atherosclerosis. Clonal deletion in the polyclonal thymocyte population is also perturbed. Distinct age-associated defects in the generation of antigen-specific T cells include a conspicuous decline in generation of T cells recognizing an immunodominant influenza epitope. Increased catalase activity delays thymic atrophy, and here, we show that it mitigates declining production of influenza-specific T cells and their frequency in lung after infection, but does not reverse declines in TRA expression or efficient negative selection. These results reveal important considerations for strategies to restore thymic function.
    Keywords:  autoimmunity; central tolerance; immunosenescence; thymus
  23. FASEB J. 2022 Mar;36(3): e22194
      The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo-obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin-binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG-132, indicating that there might be proteasome-independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1-mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1-mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability.
    Keywords:   leiomodin1 (LMOD1) ; actin nucleation; gene mutation; pediatric intestinal pseudo-obstruction; second actin-binding site
  24. JCI Insight. 2022 Feb 15. pii: e153672. [Epub ahead of print]
      Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by the accumulation of myofibroblasts and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-seq on lung fibroblasts isolated from young and aged mice during the early resolution phase post-bleomycin injury. We discovered that relative to injured young fibroblasts, injured aged fibroblasts exhibited a pro-fibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified pro-viral integration site of Moloney murine leukemia virus 1 (PIM1) and its target Nuclear Factor of Activated T Cells-1 (NFATc1) as putative drivers of the sustained pro-fibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts in vitro potentiated their fibrogenic activation in a NFATc1-dependent manner. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Inhibition of PIM1 signaling in IPF lung explants ex vivo inhibited pro-survival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.
    Keywords:  Aging; Fibrosis; Pulmonology
  25. Nat Cell Biol. 2022 Feb;24(2): 194-204
      During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals.
  26. Cell. 2022 Feb 16. pii: S0092-8674(22)00075-7. [Epub ahead of print]
      Fungal communities (the mycobiota) are an integral part of the gut microbiota, and the disruption of their integrity contributes to local and gut-distal pathologies. Yet, the mechanisms by which intestinal fungi promote homeostasis remain unclear. We characterized the mycobiota biogeography along the gastrointestinal tract and identified a subset of fungi associated with the intestinal mucosa of mice and humans. Mucosa-associated fungi (MAF) reinforced intestinal epithelial function and protected mice against intestinal injury and bacterial infection. Notably, intestinal colonization with a defined consortium of MAF promoted social behavior in mice. The gut-local effects on barrier function were dependent on IL-22 production by CD4+ T helper cells, whereas the effects on social behavior were mediated through IL-17R-dependent signaling in neurons. Thus, the spatial organization of the gut mycobiota is associated with host-protective immunity and epithelial barrier function and might be a driver of the neuroimmune modulation of mouse behavior through complementary Type 17 immune mechanisms.
    Keywords:  Th17; fungal consortia; gut-brain axis; intestinal barrier; microbiota biogeography; mycobiome; mycobiota; neuroimmune interactions; social behavior
  27. Cancer Cell. 2022 Feb 14. pii: S1535-6108(22)00011-3. [Epub ahead of print]40(2): 120-122
      In this issue of Cancer Cell, Aftab et al. identify a pro-inflammatory cytokine, IL-33, that is released as a chemoattractant for type 2 immune cells in response to the intratumoral mycobiome. Depletion of fungi or deletion of IL-33 in cancer cells significantly decreases pancreatic ductal adenocarcinoma (PDAC) tumor progression and increases survival.
  28. Sci Immunol. 2022 Feb 18. 7(68): eabi9768
      Despite IL-9 functioning as a pleiotropic cytokine in mucosal environments, the IL-9-responsive cell repertoire is still not well defined. Here, we found that IL-9 mediates proallergic activities in the lungs by targeting lung macrophages. IL-9 inhibits alveolar macrophage expansion and promotes recruitment of monocytes that develop into CD11c+ and CD11c- interstitial macrophage populations. Interstitial macrophages were required for IL-9-dependent allergic responses. Mechanistically, IL-9 affected the function of lung macrophages by inducing Arg1 activity. Compared with Arg1-deficient lung macrophages, Arg1-expressing macrophages expressed greater amounts of CCL5. Adoptive transfer of Arg1+ lung macrophages but not Arg1- lung macrophages promoted allergic inflammation that Il9r-/- mice were protected against. In parallel, the elevated expression of IL-9, IL-9R, Arg1, and CCL5 was correlated with disease in patients with asthma. Thus, our study uncovers an IL-9/macrophage/Arg1 axis as a potential therapeutic target for allergic airway inflammation.
  29. J Clin Invest. 2022 Feb 15. pii: e156507. [Epub ahead of print]
      Targeted monoclonal antibody (mAb) therapies show great promise for the treatment of transplant rejection and autoimmune diseases by inducing more specific immunomodulatory effects than broadly immunosuppressive drugs routinely used. We recently described the therapeutic advantage of targeting CD45RC, expressed at high levels by conventional T cells (Tconv, CD45RChigh), their precursors and terminally differentiated T (TEMRA) cells, but not by regulatory T cells (Tregs, CD45RClow/-). We demonstrated efficacy of anti-CD45RC mAb treatment in transplantation but its potential has not been examined in autoimmune diseases. APECED is a rare genetic syndrome caused by loss-of-function mutations of the key central tolerance mediator, autoimmune regulator (AIRE) leading to abnormal auto-reactive T cell responses and autoantibodies production. Herein, we showed that, in a rat model of APECED syndrome, anti-CD45RC mAb was effective both as prevention and treatment of autoimmune manifestations and inhibited autoantibody development. Anti-CD45RC mAb intervention depleted CD45RChigh T cells, inhibited CD45RChigh B cells, and restored the Treg/Tconv ratio and the altered Tregs transcriptomic profile. In APECED patients, CD45RC was significantly increased in peripheral blood T cells and lesioned organs from APECED patients were infiltrated by CD45RChigh cells. Our observations highlight the potential role for CD45RChigh cells in the pathogenesis of experimental and human APECED syndrome and the potential of anti-CD45RC antibody treatment.
    Keywords:  Autoimmune diseases; Autoimmunity; Immunotherapy; Therapeutics; Tolerance
  30. Front Immunol. 2022 ;13 811131
      Monoclonal antibodies targeting immune checkpoints improved clinical outcome of patients with malignant melanoma. However, the mechanisms are not fully elucidated. Since immune check-point receptors are also expressed by helper innate lymphoid cells (ILCs), we investigated the capability of immune checkpoints inhibitors to modulate ILCs in metastatic melanoma patients as well as melanoma cells effects on ILC functions. Here, we demonstrated that, compared to healthy donors, patients showed a higher frequency of total peripheral ILCs, lower percentages of CD117+ ILC2s and CD117+ ILCs as well as higher frequencies of CD117- ILCs. Functionally, melanoma patients also displayed an impaired TNFα secretion by CD117- ILCs and CD117+ ILCs. Nivolumab therapy reduced the frequency of total peripheral ILCs but increased the percentage of CD117- ILC2s and enhanced the capability of ILC2s and CD117+ ILCs to secrete IL-13 and TNFα, respectively. Before Nivolumab therapy, high CCL2 serum levels were associated with longer Overall Survival and Progression Free Survival. After two months of treatment, CD117- ILC2s frequency as well as serum concentrations of IL-6, CXCL8 and VEGF negatively correlated with both the parameters. Moreover, melanoma cells boosted TNFα production in all ILC subsets and increased the number of IL-13 producing ILC2s in vitro. Our work shows for the first time that PD-1 blockade is able to affect ILCs proportions and functions in melanoma patients and that a specific subpopulation is associated with the therapy response.
    Keywords:  cytokines; immune checkpoints inhibitors; innate lymphoid cells; melanoma; nivolumab
  31. Circulation. 2022 02 15. 145(7): 531-548
      BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown.METHODS: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcus pyogenes proteomes were used to identify mimic epitopes.
    RESULTS: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and S pyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro.
    CONCLUSIONS: ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.
    Keywords:  T-lymphocytes; autoimmunity; heart valve diseases; rheumatic diseases
  32. J Biol Chem. 2022 Feb 11. pii: S0021-9258(22)00133-8. [Epub ahead of print] 101693
      If a coronary blood vessel is occluded and the neighboring cardiomyocytes deprived of oxygen, subsequent reperfusion of the ischemic tissue can lead to oxidative damage due to excessive generation of reactive oxygen species. Cardiomyocytes and their mitochondria are the main energy producers and consumers of the heart and their metabolic changes during ischemia seem to be a key driver of reperfusion injury. Here, we hypothesized that tracking changes in cardiomyocyte metabolism, such as oxygen and ATP concentrations, would help in identifying points of metabolic failure during ischemia and reperfusion. To track some of these changes continuously from the onset of ischemia through reperfusion, we developed a system of differential equations representing the chemical reactions involved in the production and consumption of 67 molecular species. This model was validated and used to identify conditions present during periods of critical transition in ischemia and reperfusion that could lead to oxidative damage. These simulations identified a range of oxygen concentrations that lead to reverse mitochondrial electron transport at complex I of the respiratory chain and a spike in mitochondrial membrane potential, which are key suspects in the generation of reactive oxygen species at the onset of reperfusion. Our model predicts that a short initial reperfusion treatment with reduced oxygen content (5% of physiological levels) could reduce the cellular damage from both of these mechanisms. This model should serve as an open-source platform to test ideas for treatment of the ischemia reperfusion process by following the temporal evolution of molecular concentrations in the cardiomyocyte.
    Keywords:  biochemical modeling; cardiomyocyte metabolism; computational modeling; ischemia/reperfusion injury; mitochondrial membrane complexes; oxidative damage; reactive oxygen species
  33. J Exp Med. 2022 Mar 07. pii: e20220012. [Epub ahead of print]219(3):
      Liu et al. (2022. J. Exp. Med. in this issue show that T cell-independent germinal centers (GCs) can produce long-lived memory and plasma cell output. This may help explain how polysaccharide antigens provide long-term protection.
  34. Nat Biotechnol. 2022 Feb 14.
      Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.
  35. Nat Rev Drug Discov. 2022 Feb 16.
      Astrocytes are abundant glial cells in the central nervous system (CNS) that perform diverse functions in health and disease. Astrocyte dysfunction is found in numerous diseases, including multiple sclerosis, Alzheimer disease, Parkinson disease, Huntington disease and neuropsychiatric disorders. Astrocytes regulate glutamate and ion homeostasis, cholesterol and sphingolipid metabolism and respond to environmental factors, all of which have been implicated in neurological diseases. Astrocytes also exhibit significant heterogeneity, driven by developmental programmes and stimulus-specific cellular responses controlled by CNS location, cell-cell interactions and other mechanisms. In this Review, we highlight general mechanisms of astrocyte regulation and their potential as therapeutic targets, including drugs that alter astrocyte metabolism, and therapies that target transporters and receptors on astrocytes. Emerging ideas, such as engineered probiotics and glia-to-neuron conversion therapies, are also discussed. We further propose a concise nomenclature for astrocyte subsets that we use to highlight the roles of astrocytes and specific subsets in neurological diseases.
  36. Sci Adv. 2022 Feb 18. 8(7): eabf7262
      Mutations in tubulins cause distinct neurodevelopmental and degenerative diseases termed "tubulinopathies"; however, little is known about the functional requirements of tubulins or how mutations cause cell-specific pathologies. Here, we identify a mutation in the gene Tubb4a that causes degeneration of cerebellar granule neurons and myelination defects. We show that the neural phenotypes result from a cell type-specific enrichment of a dominant mutant form of Tubb4a relative to the expression other β-tubulin isotypes. Loss of Tubb4a function does not underlie cellular pathology but is compensated by the transcriptional up-regulation of related tubulin genes in a cell type-specific manner. This work establishes that the expression of a primary tubulin mutation in mature neurons is sufficient to promote cell-autonomous cell death, consistent with a causative association of microtubule dysfunction with neurodegenerative diseases. These studies provide evidence that mutations in tubulins cause specific phenotypes based on expression ratios of tubulin isotype genes.
  37. Nat Rev Immunol. 2022 Feb 15.
      Cohesin mediates chromatin loop formation across the genome by extruding chromatin between convergently oriented CTCF-binding elements. Recent studies indicate that cohesin-mediated loop extrusion in developing B cells presents immunoglobulin heavy chain (Igh) variable (V), diversity (D) and joining (J) gene segments to RAG endonuclease through a process referred to as RAG chromatin scanning. RAG initiates V(D)J recombinational joining of these gene segments to generate the large number of different Igh variable region exons that are required for immune responses to diverse pathogens. Antigen-activated mature B cells also use chromatin loop extrusion to mediate the synapsis, breakage and end joining of switch regions flanking Igh constant region exons during class-switch recombination, which allows for the expression of different antibody constant region isotypes that optimize the functions of antigen-specific antibodies to eliminate pathogens. Here, we review recent advances in our understanding of chromatin loop extrusion during V(D)J recombination and class-switch recombination at the Igh locus.
  38. Diabetes. 2022 Feb 15. pii: db210801. [Epub ahead of print]
      Dysregulation of extracellular matrix proteins in obese adipose tissue (AT) induces systemic insulin resistance. The metabolic roles of type VI collagen and its cleavage peptide endotrophin in obese AT are well established. However, the mechanisms regulating endotrophin generation remain elusive. Herein, we identified that several endotrophin-containing peptides (pre-endotrophins) were generated from the COL6A3 chain in a stepwise manner for the efficient production of mature endotrophin, partly through the action of hypoxia-induced matrix metalloproteinases (MMPs), including MMP2, MMP9, and MMP16. Hypoxia is an upstream regulator of COL6A3 expression and the proteolytic processing that regulates endotrophin generation. Hypoxia-inducible factor 1α (HIF1α) and the hypoxia-associated suppression of microRNA (miR)-29 cooperatively control the levels of COL6A3 and MMPs, which are responsible for endotrophin generation in hypoxic ATs. Adipocyte-specific Hif1a knock-out (APN-HIF1αKO) mice fed a chronic high-fat diet exhibited the significant amelioration of both local fibro-inflammation in AT and systemic insulin resistance compared to the control littermates, partly through the inhibition of endotrophin generation. Strikingly, adenovirus-mediated miR-29 overexpression in the ATs of APN-HIF1αKO mice in obesity significantly decreased endotrophin levels, suggesting that miR-29, combined with HIF1α inhibition in AT, could be a promising therapeutic strategy for treating obesity and related metabolic diseases.
  39. Cell. 2022 Feb 02. pii: S0092-8674(22)00135-0. [Epub ahead of print]
      SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.
    Keywords:  COVID-19; anosmia; nuclear architecture
  40. Mucosal Immunol. 2022 Feb 18.
      Although eosinophils are important contributors to mucosal immune responses, mechanisms that regulate their accumulation in mucosal-associated lymphoid tissues remain ill-defined. Combining bone marrow chimeras and pharmacological inhibition approaches, here we find that lymphotoxin-beta receptor (LTβR) signaling during the neonatal period is required for the accumulation of eosinophils in the mesenteric lymph nodes (MLN) during an enteric viral infection in adult male and female mice. We demonstrate that MLN stromal cells express genes that are important for eosinophil migration and survival, such as Ccl-11 (eotaxin-1), Ccl7, Ccl9, and Cxcl2, and that expression of most of these genes is downregulated as a consequence of neonatal LTβR blockade. We also find that neonatal LTβR signaling is required for the generation of a rotavirus-specific IgA antibody response in the adult MLN, but eosinophils are dispensable for this response. Collectively, our studies reveal a role for neonatal LTβR signaling in regulating eosinophil numbers in the adult MLN.
  41. Front Immunol. 2021 ;12 792813
      Integrins in effector T cells are crucial for cell adhesion and play a central role in cell-mediated immunity. Leukocyte adhesion deficiency (LAD) type III, a genetic condition that can cause death in early childhood, highlights the importance of integrin/kindlin interactions for immune system function. A TTT/AAA mutation in the cytoplasmic domain of the β2 integrin significantly reduces kindlin-3 binding to the β2 tail, abolishes leukocyte adhesion to intercellular adhesion molecule 1 (ICAM-1), and decreases T cell trafficking in vivo. However, how kindlin-3 affects integrin function in T cells remains incompletely understood. We present an examination of LFA-1/ICAM-1 bonds in both wild-type effector T cells and those with a kindlin-3 binding site mutation. Adhesion assays show that effector T cells carrying the kindlin-3 binding site mutation display significantly reduced adhesion to the integrin ligand ICAM-1. Using optical trapping, combined with back focal plane interferometry, we measured a bond rupture force of 17.85 ±0.63 pN at a force loading rate of 30.21 ± 4.35 pN/s, for single integrins expressed on wild-type cells. Interestingly, a significant drop in rupture force of bonds was found for TTT/AAA-mutant cells, with a measured rupture force of 10.08 ± 0.88pN at the same pulling rate. Therefore, kindlin-3 binding to the cytoplasmic tail of the β2-tail directly affects catch bond formation and bond strength of integrin-ligand bonds. As a consequence of this reduced binding, CD8+ T cell activation in vitro is also significantly reduced.
    Keywords:  ICAM-1; LFA-1; T cell; bond strength; kindlin-3
  42. Cell Rep. 2022 02 15. pii: S2211-1247(22)00100-0. [Epub ahead of print]38(7): 110379
      Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.
    Keywords:  human pluripotent stem cell; intestinal organoid; intestine; organoid; serosal mesothelium; suspension culture
  43. Nature. 2022 Feb 14.
      The human brain vasculature is of great medical importance: its dysfunction causes disability and death1, and the specialized structure it forms-the blood-brain barrier-impedes the treatment of nearly all brain disorders2,3. Yet so far, we have no molecular map of the human brain vasculature. Here we develop vessel isolation and nuclei extraction for sequencing (VINE-seq) to profile the major vascular and perivascular cell types of the human brain through 143,793 single-nucleus transcriptomes from 25 hippocampus and cortex samples of 9 individuals with Alzheimer's disease and 8 individuals with no cognitive impairment. We identify brain-region- and species-enriched genes and pathways. We reveal molecular principles of human arteriovenous organization, recapitulating a gradual endothelial and punctuated mural cell continuum. We discover two subtypes of human pericytes, marked by solute transport and extracellular matrix (ECM) organization; and define perivascular versus meningeal fibroblast specialization. In Alzheimer's disease, we observe selective vulnerability of ECM-maintaining pericytes and gene expression patterns that implicate dysregulated blood flow. With an expanded survey of brain cell types, we find that 30 of the top 45 genes that have been linked to Alzheimer's disease risk by genome-wide association studies (GWASs) are expressed in the human brain vasculature, and we confirm this by immunostaining. Vascular GWAS genes map to endothelial protein transport, adaptive immune and ECM pathways. Many are microglia-specific in mice, suggesting a partial evolutionary transfer of Alzheimer's disease risk. Our work uncovers the molecular basis of the human brain vasculature, which will inform our understanding of overall brain health, disease and therapy.
  44. Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2115912119. [Epub ahead of print]119(8):
      Thin endometrium has been widely recognized as a critical cause of infertility, recurrent pregnancy loss, and placental abnormalities; however, access to effective treatment is a formidable challenge due to the rudimentary understanding of the pathogenesis of thin endometrium. Here, we profiled the transcriptomes of human endometrial cells at single-cell resolution to characterize cell types, their communications, and the underlying mechanism of endometrial growth in normal and thin endometrium during the proliferative phase. Stromal cells were the most abundant cell type in the endometrium, with a subpopulation of proliferating stromal cells whose cell cycle signaling pathways were compromised in thin endometrium. Both single-cell RNA sequencing and experimental verification revealed cellular senescence in the stroma and epithelium accompanied by collagen overdeposition around blood vessels. Moreover, decreased numbers of macrophages and natural killer cells further exacerbated endometrial thinness. In addition, our results uncovered aberrant SEMA3, EGF, PTN, and TWEAK signaling pathways as causes for the insufficient proliferation of the endometrium. Together, these data provide insight into therapeutic strategies for endometrial regeneration and growth to treat thin endometrium.
    Keywords:  cell proliferation; cellular senescence; single-cell sequencing; thin endometrium
  45. JCI Insight. 2022 Feb 15. pii: e157906. [Epub ahead of print]
      BACKGROUND: NAFLD affects 25-30% of the US and European populations and is associated with insulin resistance (IR), T2D, increased cardiovascular risk and is defined by hepatic triglyceride content (HTG) > 5.56%. However, it is unknown whether HTG content less than 5.56% is associated with cardiometabolic risk factors and whether there are ethnic [Asian Indian (AI) vs. non-Asian Indian (non-AI)] and/or gender differences in these parameters in lean individuals.METHODS: We prospectively recruited 2,331 individuals and measured HTG, using 1H MRS, and plasma concentrations of triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol, and uric acid. Insulin sensitivity was assessed using HOMA-IR and the Matsuda Insulin Sensitivity Index (ISI).
    RESULTS: The 95th percentile for HTG in lean non-AI individuals was 1.85%. Plasma insulin, triglycerides, total cholesterol, LDL cholesterol and uric acid concentrations were increased and HDL decreased in individuals with HTG content > 1.85% and ≤ 5.56% compared to those individuals with HTG content ≤ 1.85% and was associated with increased IR. Mean HTG was lower in lean non-AI women compared to lean non-AI men, whereas lean AI men and women had a 40-100% increase in HTG when compared to non-AI men and women which was associated with increased cardiometabolic risk factors.
    CONCLUSIONS: We found that the 95th percentile of HTG in lean non-AI individuals was 1.85% and that HTG concentrations above this threshold were associated with IR and cardiovascular risk factors. Premenopausal women are protected from these changes whereas young lean AI men and women manifest increased HTG content and associated cardiometabolic risk factors.
    FUNDING: Supported by grants from the United States Department of Health and Human Resources (NIH/NIDDK): R01 DK113984, P30 DK45735, U24 DK59635 and UL1 RR024139 and the Novo Nordisk Foundation (NNF18CC0034900).
    Keywords:  Glucose metabolism; Hepatology; Insulin; Metabolism; Obesity
  46. Nat Commun. 2022 Feb 15. 13(1): 874
      IL-18 is emerging as an IL-22-induced and epithelium-derived cytokine which contributes to host defence against intestinal infection and inflammation. In contrast to its known role in Goblet cells, regulation of barrier function at the molecular level by IL-18 is much less explored. Here we show that IL-18 is a bona fide IL-22-regulated gate keeper for intestinal epithelial barrier. IL-22 promotes crypt immunity both via induction of phospho-Stat3 binding to the Il-18 gene promoter and via Il-18 independent mechanisms. In organoid culture, while IL-22 primarily increases organoid size and inhibits expression of stem cell genes, IL-18 preferentially promotes organoid budding and induces signature genes of Lgr5+ stem cells via Akt-Tcf4 signalling. During adherent-invasive E. coli (AIEC) infection, systemic administration of IL-18 corrects compromised T-cell IFNγ production and restores Lysozyme+ Paneth cells in Il-22-/- mice, but IL-22 administration fails to restore these parameters in Il-18-/- mice, thereby placing IL-22-Stat3 signalling upstream of the IL-18-mediated barrier defence function. IL-18 in return regulates Stat3-mediated anti-microbial response in Paneth cells, Akt-Tcf4-triggered expansion of Lgr5+ stem cells to facilitate tissue repair, and AIEC clearance by promoting IFNγ+ T cells.
  47. Nat Cell Biol. 2022 Feb;24(2): 181-193
      The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.
  48. Front Immunol. 2022 ;13 838719
      The underlying mechanisms of thymocyte development and lineage determination remain incompletely understood, and the emerging evidences demonstrated that RNA binding proteins (RBPs) are deeply involved in governing T cell fate in thymus. Serine/arginine-rich splicing factor 1 (SRSF1), as a classical splicing factor, is a pivotal RBP for gene expression in various biological processes. Our recent study demonstrated that SRSF1 plays essential roles in the development of late thymocytes by modulating the T cell regulatory gene networks post-transcriptionally, which are critical in response to type I interferon signaling for supporting thymocyte maturation. Here, we report SRSF1 also contributes to the determination of the CD8+ T cell fate. By specific ablation of SRSF1 in CD4+CD8+ double positive (DP) thymocytes, we found that SRSF1 deficiency impaired the maturation of late thymocytes and diminished the output of both CD4+ and CD8+ single positive T cells. Interestingly, the ratio of mature CD4+ to CD8+ cells was notably altered and more severe defects were exhibited in CD8+ lineage than those in CD4+ lineage, reflecting the specific function of SRSF1 in CD8+ T cell fate decision. Mechanistically, SRSF1-deficient cells downregulate their expression of Runx3, which is a crucial transcriptional regulator in sustaining CD8+ single positive (SP) thymocyte development and lineage choice. Moreover, forced expression of Runx3 partially rectified the defects in SRSF1-deficient CD8+ thymocyte maturation. Thus, our data uncovered the previous unknown role of SRSF1 in establishment of CD8+ cell identity.
    Keywords:  CD8+ T cell; Runx3; SRSF1; development ; lineage choice; thymocyte
  49. Immunity. 2022 Feb 12. pii: S1074-7613(22)00082-6. [Epub ahead of print]
      The generation of memory B cells and plasma cells is complex and involves inputs from the microenvironment, notably from T follicular helper cells. In last month's issue of Immunity, Yeh et al. and Song et al. refine our understanding of the B cell intrinsic and extrinsic requirements to generate effective humoral immunity in response to foreign antigens.
  50. Cell Rep. 2022 02 15. pii: S2211-1247(22)00110-3. [Epub ahead of print]38(7): 110389
      Liver sinusoidal endothelial cells (LSECs) are liver-resident antigen (cross)-presenting cells that generate memory CD8 T cells, but metabolic properties of LSECs and LSEC-primed CD8 T cells remain understudied. Here, we report that high-level mitochondrial respiration and constitutive low-level glycolysis support LSEC scavenger and sentinel functions. LSECs fail to increase glycolysis and co-stimulation after TLR4 activation, indicating absence of metabolic and functional maturation compared with immunogenic dendritic cells. LSEC-primed CD8 T cells show a transient burst of oxidative phosphorylation and glycolysis. Mechanistically, co-stimulatory IL-6 signaling ensures high FOXO1 expression in LSEC-primed CD8 T cells, curtails metabolic activity associated with T cell activation, and is indispensable for T cell functionality after re-activation. Thus, distinct immunometabolic features characterize non-immunogenic LSECs compared with immunogenic dendritic cells and LSEC-primed CD8 T cells with memory features compared with effector CD8 T cells. This reveals local features of metabolism and function of T cells in the liver.
    Keywords:  glycolysis; immune cell metabolism; liver immune tolerance; memory T cells; mitochondrial respiration; non-professional antigen-presenting cells
  51. Sci Rep. 2022 Feb 14. 12(1): 2444
      Cell cycle associated protein 1 (Caprin1) is an RNA-binding protein that can regulate the cellular post-transcriptional response to stress. It is a component of both stress granules and neuronal RNA granules and is implicated in neurodegenerative disease, synaptic plasticity and long-term memory formation. Our previous work suggested that Caprin1 also plays a role in the response of the cochlea to stress. Here, targeted inner ear-deletion of Caprin1 in mice leads to an early onset, progressive hearing loss. Auditory brainstem responses from Caprin1-deficient mice show reduced thresholds, with a significant reduction in wave-I amplitudes compared to wildtype. Whilst hair cell structure and numbers were normal, the inner hair cell-spiral ganglion neuron (IHC-SGN) synapse revealed abnormally large post-synaptic GluA2 receptor puncta, a defect consistent with the observed wave-I reduction. Unlike wildtype mice, mild-noise-induced hearing threshold shifts in Caprin1-deficient mice did not recover. Oxidative stress triggered TIA-1/HuR-positive stress granule formation in ex-vivo cochlear explants from Caprin1-deficient mice, showing that stress granules could still be induced. Taken together, these findings suggest that Caprin1 plays a key role in maintenance of auditory function, where it regulates the normal status of the IHC-SGN synapse.
  52. FASEB J. 2022 Mar;36(3): e22212
      Leucine-rich glioma-inactivated protein 1 (LGI1) is known to play a key role in autosomal dominant lateral temporal lobe epilepsy (ADLTE). The ADLTE is an inherited disease characterized by focal seizures with distinctive auditory or aphasic symptoms. A large number of mutations on the Lgi1 gene have been reported and are believed to be the genetic cause for ADLTE. We identified a novel missense mutation, c.152A>G (p.Asp51Gly), on Lgi1 from a Chinese ADLTE patient who manifests locomotor imbalance and white matter reduction. However, it remains unknown how mutant LGI1 causes white matter abnormalities at molecular and cellular levels. Here, we generated a knock-in mouse bearing this Lgi1 mutation. We found that Lgi1D51G / D51G mice exhibited impaired defective white matter and motor coordination. We observed that Lgi1D51G / D51G mice displayed a reduced number of mature oligodendrocytes (OLs) and deficient OL differentiation in the white matter. However, the population of oligodendrocyte precursor cells was not affected in Lgi1D51G / D51G mice. Mechanistically, we showed that the Lgi1D51G mutation resulted in altered mTOR signaling and led to decreased levels of Sox10. Given that Sox10 is a key transcriptional factor to control OL differentiation, our results strongly suggest that the Lgi1D51G mutation may cause white matter abnormalities via inhibiting Sox10-dependent OL differentiation and myelination in the central nervous system.
    Keywords:  LGI1; Sox10; myelination; oligodendrocyte differentiation; white matter
  53. Nat Commun. 2022 Feb 15. 13(1): 878
      In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.
  54. Nat Chem Biol. 2022 Feb 14.
      Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.
  55. Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2118535119. [Epub ahead of print]119(8):
      Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5+ ILC2 responses, and induced a dominant IL-13+ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ-deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR+ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.
    Keywords:  IFN-γ; ILCs; PB1-F2; T cells; influenza
  56. Science. 2022 Feb 18. 375(6582): eabc4203
      Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We find that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.
  57. Aging Cell. 2022 Feb 16. e13558
      Age is a risk factor for numerous diseases, including neurodegenerative diseases, cancers, and diabetes. Loss of protein homeostasis is a central hallmark of aging. Activation of the endoplasmic reticulum unfolded protein response (UPRER ) includes changes in protein translation and membrane lipid synthesis. Using stable isotope labeling, a flux "signature" of the UPRER in vivo in mouse liver was developed by inducing ER stress with tunicamycin and measuring rates of both proteome-wide translation and de novo lipogenesis. Several changes in protein synthesis across ontologies were noted with age, including a more dramatic suppression of translation under ER stress in aged mice as compared with young mice. Binding immunoglobulin protein (BiP) synthesis rates and mRNA levels were increased more in aged than young mice. De novo lipogenesis rates decreased under ER stress conditions in aged mice, including both triglyceride and phospholipid fractions. In young mice, a significant reduction was seen only in the triglyceride fraction. These data indicate that aged mice have an exaggerated metabolic flux response to ER stress, which may indicate that aging renders the UPRER less effective in resolving proteotoxic stress.
    Keywords:  aging; de novo lipogenesis; endoplasmic reticulum; proteome dynamics; proteomics; unfolded protein response
  58. Cell Rep. 2022 02 15. pii: S2211-1247(22)00086-9. [Epub ahead of print]38(7): 110365
      AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy.
    Keywords:  AMPK; energy homeostasis; kinase; lysosome; mTORC1; metabolic signaling; phosphorylation
  59. Nat Cell Biol. 2022 Feb 17.
      Metabolic reprogramming is central to oncogene-induced tumorigenesis by providing the necessary building blocks and energy sources, but how oncogenic signalling controls metabolites that play regulatory roles in driving cell proliferation and tumour growth is less understood. Here we show that oncogene YAP/TAZ promotes polyamine biosynthesis by activating the transcription of the rate-limiting enzyme ornithine decarboxylase 1. The increased polyamine levels, in turn, promote the hypusination of eukaryotic translation factor 5A (eIF5A) to support efficient translation of histone demethylase LSD1, a transcriptional repressor that mediates a bulk of YAP/TAZ-downregulated genes including tumour suppressors in YAP/TAZ-activated cells. Accentuating the importance of the YAP/TAZ-polyamine-eIF5A hypusination-LSD1 axis, inhibiting polyamine biosynthesis or LSD1 suppressed YAP/TAZ-induced cell proliferation and tumour growth. Given the frequent upregulation of YAP/TAZ activity and polyamine levels in diverse cancers, our identification of YAP/TAZ as an upstream regulator and LSD1 as a downstream effector of the oncometabolite polyamine offers a molecular framework in which oncogene-induced metabolic and epigenetic reprogramming coordinately drives tumorigenesis, and suggests potential therapeutic strategies in YAP/TAZ- or polyamine-dependent human malignancies.
  60. PLoS Genet. 2022 Feb 16. 18(2): e1010049
      The epigenetic landscape of a cell frequently changes in response to fluctuations in nutrient levels, but the mechanistic link is not well understood. In fission yeast, the JmjC domain protein Epe1 is critical for maintaining the heterochromatin landscape. While loss of Epe1 results in heterochromatin expansion, overexpression of Epe1 leads to defective heterochromatin. Through a genetic screen, we found that mutations in genes of the cAMP signaling pathway suppress the heterochromatin defects associated with Epe1 overexpression. We further demonstrated that the activation of Pka1, the downstream effector of cAMP signaling, is required for the efficient translation of epe1+ mRNA to maintain Epe1 overexpression. Moreover, inactivation of the cAMP-signaling pathway, either through genetic mutations or glucose deprivation, leads to the reduction of endogenous Epe1 and corresponding heterochromatin changes. These results reveal the mechanism by which the cAMP signaling pathway regulates heterochromatin landscape in fission yeast.