DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations.

BACKGROUND: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. OBJECTIVE: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. METHODS: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. RESULTS: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. CONCLUSIONS: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.

Mycobacteria exploit nitric oxide-induced transformation of macrophages into permissive giant cells.

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so-called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double-edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO-induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria-infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.

When cilia go bad: cilia defects and ciliopathies.

Defects in the function of cellular organelles such as peroxisomes, lysosomes and mitochondria are well-known causes of human diseases. Recently, another organelle has also been added to this list. Cilia--tiny hair-like organelles attached to the cell surface--are located on almost all polarized cell types of the human body and have been adapted as versatile tools for various cellular functions, explaining why cilia-related disorders can affect many organ systems. Several molecular mechanisms involved in cilia-related disorders have been identified that affect the structure and function of distinct cilia types.

Plasma cell deficiency in human subjects with heterozygous mutations in Sec61 translocon alpha 1 subunit (SEC61A1).

BACKGROUND: Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. OBJECTIVE: We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. METHODS: Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. RESULTS: We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On in vitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. CONCLUSION: We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding our knowledge about the pathogenesis of antibody deficiencies.

Haploinsufficiency of the NF-κB1 Subunit p50 in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID), characterized by recurrent infections, is the most prevalent symptomatic antibody deficiency. In ∼90% of CVID-affected individuals, no genetic cause of the disease has been identified. In a Dutch-Australian CVID-affected family, we identified a NFKB1 heterozygous splice-donor-site mutation (c.730+4A>G), causing in-frame skipping of exon 8. NFKB1 encodes the transcription-factor precursor p105, which is processed to p50 (canonical NF-κB pathway). The altered protein bearing an internal deletion (p.Asp191_Lys244delinsGlu; p105ΔEx8) is degraded, but is not processed to p50ΔEx8. Altered NF-κB1 proteins were also undetectable in a German CVID-affected family with a heterozygous in-frame exon 9 skipping mutation (c.835+2T>G) and in a CVID-affected family from New Zealand with a heterozygous frameshift mutation (c.465dupA) in exon 7. Given that residual p105 and p50­translated from the non-mutated alleles­were normal, and altered p50 proteins were absent, we conclude that the CVID phenotype in these families is caused by NF-κB1 p50 haploinsufficiency.

Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency.

BACKGROUND: Most patients with common variable immunodeficiency (CVID) present with severely reduced switched memory B-cell counts, and some display an increase of CD21low B-cell counts (CVID 21low), whereas others do not (CVID 21norm). Altered B-cell receptor (BCR) signaling might contribute to the defective memory formation observed in patients with CVID. OBJECTIVE: We sought to investigate canonical nuclear factor of κ light chain (NF-κB) signaling in B cells from patients with CVID as a central pathway in B-cell differentiation. METHODS: Degradation of inhibitor of κBα (IκBα) and p65 phosphorylation, nuclear translocation of p65, and regulation of target genes and cell function were investigated after different modes of B-cell stimulation. RESULTS: BCR-mediated canonical NF-κB signaling was impaired in all mature naive CVID-derived B cells. This impairment was more profound in naive B cells from CVID 21low patients than CVID 21norm patients and most pronounced in CD21low B cells. The signaling defect translated into reduced induction of Bcl-xL and IκBα, 2 bona fide target genes of the canonical NF-κB pathway. CD40 ligand- and Toll-like receptor 9-mediated signaling were less strongly altered. Signaling in CD21low B cells but not CD21+ B cells of patients with HIV was similarly affected. CONCLUSION: Combined with the previous description of disturbed Ca2+ signaling, the discovery of NF-κB signaling defects, especially in CVID 21low patients, suggests a broad underlying signaling defect affecting especially BCR-derived signals. Given the immune phenotype of monogenic defects affecting Ca2+ and NF-κB signaling, the latter is more likely to contribute to the humoral deficiency. The strongly disturbed BCR signaling of CD21low B cells is characteristic for this cell type and independent of the underlying disease.

DCLRE1C (ARTEMIS) mutations causing phenotypes ranging from atypical severe combined immunodeficiency to mere antibody deficiency.

Null mutations in genes involved in V(D)J recombination cause a block in B- and T-cell development, clinically presenting as severe combined immunodeficiency (SCID). Hypomorphic mutations in the non-homologous end-joining gene DCLRE1C (encoding ARTEMIS) have been described to cause atypical SCID, Omenn syndrome, Hyper IgM syndrome and inflammatory bowel disease-all with severely impaired T-cell immunity. By whole-exome sequencing, we investigated the molecular defect in a consanguineous family with three children clinically diagnosed with antibody deficiency. We identified perfectly segregating homozygous variants in DCLRE1C in three index patients with recurrent respiratory tract infections, very low B-cell numbers and serum IgA levels. In patients, decreased colony survival after irradiation, impaired proliferative response and reduced counts of naïve T cells were observed in addition to a restricted T-cell receptor repertoire, increased palindromic nucleotides in the complementarity determining regions 3 and long stretches of microhomology at switch junctions. Defective V(D)J recombination was complemented by wild-type ARTEMIS protein in vitro. Subsequently, homozygous or compound heterozygous DCLRE1C mutations were identified in nine patients from the same geographic region. We demonstrate that DCLRE1C mutations can cause a phenotype presenting as only antibody deficiency. This novel association broadens the clinical spectrum associated with ARTEMIS mutations. Clinicians should consider the possibility that an immunodeficiency with a clinically mild initial presentation could be a combined immunodeficiency, so as to provide appropriate care for affected patients.

NFKB1 regulates human NK cell maturation and effector functions.

NFKB1, a component of the canonical NF-κB pathway, was recently reported to be mutated in a limited number of CVID patients. CVID-associated mutations in NFKB2 (non-canonical pathway) have previously been shown to impair NK cell cytotoxic activity. Although a biological function of NFKB1 in non-human NK cells has been reported, the role of NFKB1 mutations for human NK cell biology and disease has not been investigated yet. We decided therefore to evaluate the role of monoallelic NFKB1 mutations in human NK cell maturation and functions. We show that NFKB1 mutated NK cells present impaired maturation, defective cytotoxicity and reduced IFN-γ production upon in vitro stimulation. Furthermore, human IL-2 activated NFKB1 mutated NK cells fail to up-regulate the expression of the activating marker NKp44 and show reduced proliferative capacity. These data suggest that NFKB1 plays an essential novel role for human NK cell maturation and effector functions.

The atypical cadherin Dachsous1 localizes to the base of the ciliary apparatus in airway epithelia.

Mucociliary clearance requires the distinct orientation and coordinated movement of airway cilia, which is established through planar cell polarity signaling (PCP). The atypical cadherin Dachsous1 (Dchs1) is a transmembrane protein that regulates PCP in D. melanogaster. However, little is known about Dchs1 expression and its potential role in PCP in mammalian adult tissues. Here, we show that Dchs1 is ubiquitously expressed in mouse embryos, but exhibits a highly restricted expression to lung tissues in the adult stage. Strikingly, human Dchs1 localized exclusively to the base of the ciliary apparatus in cultured human respiratory epithelial cells with differentiated motile 9 + 2 cilia. This localization could be functionally important as we observed aberrant DCHS1 mRNA expression in human non-small cell lung cancer tissue. In sum, we establish Dchs1 as a component of the membrane domain surrounding the ciliary base. This suggests a specific role of Dchs1 in PCP-dependent organization of ciliary function and a possible role in lung disease.

Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels.

BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.

Distinct localization of septin proteins to ciliary sub-compartments in airway epithelial cells.

Mucociliary clearance of the airways is accomplished by cilia-mediated laminar mucus flow along the planar epithelial surface. Maintenance of the highly specific architecture of the ciliated airway epithelium with columnar-shaped epithelial cells and tightening of the epithelial barrier is mainly attributed to the F-actin cytoskeleton. Recently, members of the highly conserved family of septin proteins have been shown to play crucial roles in ciliated tissue. These GTP-binding proteins form hetero-oligomeric complexes and assemble higher-order cytoskeletal structures such as filaments, bundles and ring-like structures such as a membrane diffusion barrier at the ciliary base. Here we analyzed the subcellular and sub-ciliary localization of various septin proteins by immunofluorescence imaging of airway epithelial cells. In addition to cytoplasmic localization we found that septins are either enriched at the apical cell cortex including the ciliary bases (septin-2, -4, -6, and -7), or show axonemal staining (septin-2, -7, -9 and -11) or specifically localize to ciliary sub-compartments (septin-8 and -9). The distinct localization of septins suggests structural functions as cytoskeletal components and as elements of the mechanical barrier at the apical cell cortex. Furthermore, the tight association of septin-8 and -9 with the ciliary compartment indicates a possible involvement in cilia-specific functions and cilia-related diseases.

Hyper-IgE syndromes: reviewing PGM3 deficiency.

PURPOSE OF REVIEW: The hyper-IgE syndromes have been recognized as a group of primary immunodeficiencies characterized by eczema, recurrent skin and lung infections, and elevated serum IgE. Recently, mutations in phosphoglucomutase 3 (encoding PGM3, which is involved in the protein glycosylation pathway) have been identified in autosomal recessive forms of hyper-IgE syndromes. RECENT FINDINGS: Autosomal recessive, hypomorphic PGM3 mutations cause a multisystem disorder, characterized by both a congenital glycosylation disease and a hyper-IgE syndrome. The reported mutations in PGM3 led to an impaired biosynthesis of UDP-GlcNAc and impaired tri-antennary and tetra-antennary N-glycan structures. Laboratory results in patients showed eosinophilia, a T-cell proliferation defect, and a reversed CD4/CD8 ratio. The impaired glycosylation in PGM3-mutant patients will not only affect proteins involved in the immune system, and thus causes a multisystem phenotype. SUMMARY: The identification of hyper-IgE syndromes-associated mutations in PGM3 provides the basis for future studies on the pathophysiology and the molecular mechanisms of eczema, IgE dysregulation, and increased susceptibility to infections.

Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins.

Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.

Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis.

Nephronophthisis (NPHP), a group of autosomal recessive cystic kidney disorders, is the most common genetic cause of progressive renal failure in children and young adults. NPHP may be associated with Leber congenital amaurosis, tapeto-retinal degeneration, cerebellar ataxia, cone-shaped epiphyses, congenital oculomotor apraxia and hepatic fibrosis. Loci associated with an infantile type of NPHP on 9q22-q31 (NPHP2), juvenile types of NPHP on chromosomes 2q12-q13 (NPHP1) and 1p36 (NPHP4) and an adolescent type of NPHP on 3q21-q22 (NPHP3) have been mapped. NPHP1 and NPHP4 have been identified, and interaction of the respective encoded proteins nephrocystin and nephrocystin-4 has been shown. Here we report the identification of NPHP3, encoding a novel 1,330-amino acid protein that interacts with nephrocystin. We describe mutations in NPHP3 in families with isolated NPHP and in families with NPHP with associated hepatic fibrosis or tapeto-retinal degeneration. We show that the mouse ortholog Nphp3 is expressed in the node, kidney tubules, retina, respiratory epithelium, liver, biliary tract and neural tissues. In addition, we show that a homozygous missense mutation in Nphp3 is probably responsible for the polycystic kidney disease (pcy) mouse phenotype. Interventional studies in the pcy mouse have shown beneficial effects by modification of protein intake and administration of methylprednisolone, suggesting therapeutic strategies for treating individuals with NPHP3.