CD45 expression discriminates waves of embryonic megakaryocytes in the mouse

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41++CD45-CD9++CD61+MPL+CD42c+ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45- until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41+CD45+ and CD41+CD45- cells. These populations, and that of CD41++CD45-CD42c+ cells, isolated from liver, differentiate in culture into CD41++CD45-CD42c+ proplatelet-bearing megakaryocytes. Also present at this time are CD41-CD45++CD11b+ cells, which produce low numbers of CD41++CD45-CD42c+ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41++CD45-CD42c+ embryo megakaryocytes both in vivo and in vitro In contrast, around 30% of adult megakaryocytes (CD41++CD45++CD9++CD42c+) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41++CD45-CD42c+ embryo megakaryocytes that can be produced from CD41+CD45- or from CD41+CD45+ cells, at difference from those from bone marrow.This work was supported by grants from the Ministerio de Ciencia e Innovacion (MICINN SAF2009-12596) and from the Ministerio de Economia y Competitividad (MINECO SAF2012-33916 and SAF2015-70880-R MINECO/FEDER). NS was the recipient of a fellowship from the Centro de Biologia Molecular Severo Ochoa (CBMSO) and IC received a fellowship from the MICINN. The CBMSO receives institutional funding from Fundacion Ramon Areces. The CNIC is supported by the MEIC and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).S

Postnatal and adult immunoglobulin repertoires of innate-like CD19(+)CD45R(lo) B Cells

The diversity in antibody repertoire relies on different B cell populations working efficiently to fulfil distinct specific functions. We recently described an innate-like CD19(+)CD45R(-/lo) (19(+)45R(lo)) cell population in postnatal unstimulated adult mice, a heterogeneous population containing cells expressing immunoglobulin M (IgM) and others behaving as differentiated mature B lymphocytes (intracytoplasmic IgG1, AID(+), Blimp-1(+)RAG2(-)). In the present study, we characterized the Ig repertoire expressed by splenic 19(+)45R(lo) cells, assuming that they would bear a restricted repertoire biased for germline rearrangements and low mutation rates similar to other innate-like cells. Sequences from 19(+)45R(lo) cells displayed a variety of V, D and J regions, and the analysis of the CDR-H3 region revealed an intermediate overall CDR-H3 length and moderate hydrophobicity. Both IgM and switched sequences of PD15 19(+)45R(lo) cells had shorter CDR-H3 region and fewer non-template N nucleotides than adult sequences, as expected for profiles that correspond to an immature phenotype. Regarding the mutation rate in the VH regions, IgG1 sequences already carried a high rate of replacement mutations at PD15, which increased further in the sequences obtained from adult mice. Moreover, statistical models suggest that a proportion of the switched sequences in adult 19(+)45R(lo) cells had experienced antigen selection, unlike other innate-like B cell compartments.We thank B. Palacios for assisting with the cloning and sequencing, A.G. de la Campa, A. Trento, I. Cuesta and L. Garcia-Albert for their help with the bioinformatics software analyses, and M.A.R. Marcos for his contribution to the original planning of the experiments. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS, MPY 1450/11) and from the Ministerio de Ciencia e Innovación (MICINN, SAF2009-12596) and Ministerio de Economia y competitividad (MINECO, SAF2012-33916). I. Cortegano received a postdoctoral fellowship from the MICINN

The TLR4-MyD88 Signaling Axis Regulates Lung Monocyte Differentiation Pathways in Response to Streptococcus pneumoniae

Streptococcus pneumoniae is the main cause of bacterial pneumonia, a condition that currently produces significant global morbidity and mortality. The initial immune response to this bacterium occurs when the innate system recognizes common motifs expressed by many pathogens, events driven by pattern recognition receptors like the Toll-like family receptors (TLRs). In this study, lung myeloid-cell populations responsible for the innate immune response (IIR) against S. pneumoniae, and their dependence on the TLR4-signaling axis, were analyzed in TLR4–/– and Myeloid-Differentiation factor-88 deficient (MyD88–/–) mice. Neutrophils and monocyte-derived cells were recruited in infected mice 3-days post-infection. Compared to wild-type mice, there was an increased bacterial load in both these deficient mouse strains and an altered IIR, although TLR4–/– mice were more susceptible to bacterial infection. These mice also developed fewer alveolar macrophages, weaker neutrophil infiltration, less Ly6Chigh monocyte differentiation and a disrupted classical and non-classical monocyte profile. The pro-inflammatory cytokine profile (CXCL1, TNF-α, IL-6, and IL-1β) was also severely affected by the lack of TLR4 and no induction of Th1 was observed in these mice. The respiratory burst (ROS production) after infection was profoundly dampened in TLR4–/– and MyD88–/– mice. These data demonstrate the complex dynamics of myeloid populations and a key role of the TLR4-signaling axis in the IIR to S. pneumoniae, which involves both the MyD88 and TRIF (Toll/IL-1R domain-containing adaptor-inducing IFN-β) dependent pathways.This work was supported by grants of Ministerio de Ciencia SAF 2015-70880-R, RTI 2018-099114-B-100, BIO 2017-82951-R, and ISCIII PI14CIII/00049.S