ABSTRACT
Recently it was shown that circulating Ly6C(+) monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C(+) monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C(+) monocytes. In this study we investigated whether Ly6C(+) monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property particularly attributed to Batf3(+) DCs. We demonstrated that Ly6C(+) monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8(+) T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C(+) monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C(+) monocytes. Overall, this study outlines two functional roles, among others, that Ly6C(+) monocytes have during an adaptive immune response.
Subject(s)
Antigens, Ly/metabolism , Monocytes/metabolism , Animals , Apoptosis/radiation effects , Basic-Leucine Zipper Transcription Factors/metabolism , Dendritic Cells/cytology , Dendritic Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Monocytes/cytology , Ovalbumin/immunology , Phagocytosis , RNA/chemistry , RNA/metabolism , Repressor Proteins/metabolism , Sequence Analysis, RNA , Spleen/cytology , Spleen/immunology , Thymocytes/cytology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Transcriptome , Ultraviolet RaysABSTRACT
The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered by an oblique lattice which consists of the S-layer protein SbsC. On SDS-polyacrylamide gels, the mature S-layer protein migrates as a single band with an apparent molecular mass of 122 kDa. During cultivation of B. stearothermophilus ATCC 12980 at 67 degrees C instead of 55 degrees C, a variant developed that had a secondary cell wall polymer identical to that of the wild-type strain, but it carried an S-layer glycoprotein that could be separated on SDS-polyacrylamide gels into four bands with apparent molecular masses of 92, 118, 150 and 175 kDa. After deglycosylation, only a single protein band with a molecular mass of 92 kDa remained. The complete nucleotide sequence encoding the protein moiety of this S-layer glycoprotein, termed SbsD, was established by PCR and inverse PCR. The sbsD gene of 2,709 bp is predicted to encode a protein of 96.2 kDa with a 30-amino-acid signal peptide. Within the 807 bp encoding the signal peptide and the N-terminal sequence (amino acids 31-269), different nucleotides for sbsD and sbsC were observed in 46 positions, but 70% of these mutations were silent, thus leading to a level of identity of 95% for the N-terminal parts. The level of identity of the remaining parts of SbsD and SbsC was below 10%, indicating that the lysine-, tyrosine- and arginine-rich N-terminal region in combination with a distinct type of secondary cell wall polymer remained conserved upon S-layer variation. The sbsD sequence encoding the mature S-layer protein cloned into the pET28a vector led to stable expression in Escherichia coli HMS174(DE3). This is the first example demonstrating that S-layer variation leads to the synthesis of an S-layer glycoprotein.