Clonal barcoding of endogenous adult hematopoietic stem cells reveals a spectrum of lineage contributions
The hierarchical model of hematopoiesis posits that self-renewing, multipotent hematopoietic stem cells (HSCs) give rise to all blood cell lineages. While this model accounts for hematopoiesis in transplant settings, its applicability to steady-state hematopoiesis remains to be clarified. Here, we used inducible clonal DNA barcoding of endogenous adult HSCs to trace their contribution to major hematopoietic cell lineages in unmanipulated animals. While the majority of barcodes were unique to a single lineage, we also observed frequent barcode sharing between multiple lineages, specifically between lymphocytes and myeloid cells. These results suggest that both single-lineage and multilineage contributions by HSCs collectively drive continuous hematopoiesis, and highlight a close relationship of myeloid and lymphoid development.
Cutting Edge: TLR2 Signaling in B Cells Promotes Autoreactivity to DNA via IL-6 Secretion
Autoantibodies to chromatin and dsDNA are a hallmark of systemic lupus erythematosus (SLE). In a mouse model of monogenic human SLE caused by DNASE1L3 deficiency, the anti-DNA response is dependent on endosomal nucleic acid-sensing TLRs TLR7 and TLR9. In this study, we report that this response also required TLR2, a surface receptor for microbial products that is primarily expressed on myeloid cells. Cell transfers into lymphopenic DNASE1L3-deficient mice showed that TLR2 was required for anti-DNA Ab production by lymphocytes. TLR2 was detectably expressed on B cells and facilitated the production of IL-6 by B cells activated in the presence of microbial products. Accordingly, treatment with broad-spectrum antibiotics or Ab-mediated blockade of IL-6 delayed the anti-DNA response in DNASE1L3-deficient mice. These studies reveal an unexpected B cell-intrinsic role of TLR2 in systemic autoreactivity to DNA, and they suggest that microbial products may synergize with self-DNA in the activation of autoreactive B cells in SLE.
Haplodeficiency of the 9p21 Tumor Suppressor Locus Causes Myeloid Disorders Driven by the Bone Marrow Microenvironment
The chromosome 9p21 locus comprises several tumor suppressor genes including MTAP, CDKN2A and CDKN2B, and its homo- or heterozygous deletion is associated with reduced survival in multiple cancer types. We report that mice with germline monoallelic deletion or induced biallelic deletion of the 9p21-syntenic locus (9p21s) developed a fatal myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN)-like disease associated with aberrant trabecular bone formation and/or fibrosis in the bone marrow (BM). Reciprocal BM transfers and conditional targeting of 9p21s suggested that the disease originates in the BM stroma. Single-cell analysis of 9p21s-deficient BM stroma revealed the expansion of chondrocyte and osteogenic precursors, reflected in increased osteogenic differentiation in vitro. It also showed reduced expression of factors maintaining hematopoietic stem/progenitor cells, including Cxcl12. Accordingly, 9p21s-deficient mice showed reduced levels of circulating Cxcl12 and concomitant upregulation of the pro-fibrotic chemokine Cxcl13 and osteogenesis- and fibrosis-related multifunctional glycoprotein Osteopontin (OPN)/Spp1. Our study highlights the potential of mutations in the BM microenvironment to drive MDS/MPN-like disease.
Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses
High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naïve T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1β secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.
Secreted mammalian DNases protect against systemic bacterial infection by digesting biofilms
Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.
Stem cell decoupling underlies impaired lymphoid development during aging
Mammalian aging is associated with multiple defects of hematopoiesis, most prominently with the impaired development of T and B lymphocytes. This defect is thought to originate in hematopoietic stem cells (HSCs) of the bone marrow, specifically due to the age-dependent accumulation of HSCs with preferential megakaryocytic and/or myeloid potential ("myeloid bias"). Here, we tested this notion using inducible genetic labeling and tracing of HSCs in unmanipulated animals. We found that the endogenous HSC population in old mice shows reduced differentiation into all lineages including lymphoid, myeloid, and megakaryocytic. Single-cell RNA sequencing and immunophenotyping (CITE-Seq) showed that HSC progeny in old animals comprised balanced lineage spectrum including lymphoid progenitors. Lineage tracing using the aging-induced HSC marker Aldh1a1 confirmed the low contribution of old HSCs across all lineages. Competitive transplantations of total bone marrow cells with genetically marked HSCs revealed that the contribution of old HSCs was reduced, but compensated by other donor cells in myeloid cells but not in lymphocytes. Thus, the HSC population in old animals becomes globally decoupled from hematopoiesis, which cannot be compensated in lymphoid lineages. We propose that this partially compensated decoupling, rather than myeloid bias, is the primary cause of the selective impairment of lymphopoiesis in older mice.
Reclassification of plasmacytoid dendritic cells as innate lymphocytes is premature [Comment]
Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells
Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.
Autoantibody-mediated impairment of DNASE1L3 activity in sporadic systemic lupus erythematosus
Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.
Human plasmacytoid dendritic cells mount a distinct antiviral response to virus-infected cells
Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, human cytomegalovirus (CMV). Human pDCs produced high amounts of type I interferon (IFN-I) when incubated with live CMV-infected fibroblasts but not with free CMV; the response involved integrin-mediated adhesion, transfer of DNA-containing virions to pDCs, and the recognition of DNA through TLR9. Compared with transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of IFN-I and IFN-III, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged, and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells critical for CMV control. Last, patients with CMV viremia harbored phenotypically activated pDCs and increased circulating IFN-I and IFN-III. Thus, recognition of live infected cells is a mechanism of virus detection by pDCs that elicits a unique antiviral immune response.