The anti-TLR4 monoclonal antibody Sa15-21 enhances inflammatory cytokine production in LPS-stimulated macrophages.

Sa15-21, a monoclonal antibody against mouse Toll-like receptor (TLR) 4, can protect mice from lipopolysaccharide (LPS)/D-galactosamine-induced acute lethal hepatitis. Herein, we investigated the molecular mechanisms underlying Sa15-21-mediated regulation of TLR4 signaling in macrophages. Results showed that Sa15-21 enhanced the production of proinflammatory cytokines and attenuated the production of anti-inflammatory cytokines in LPS-stimulated macrophages. Western blotting analysis revealed that Sa15-21 pretreatment had no effect on NF-κB and MAPK signaling in LPS-stimulated macrophages; however, Sa15-21 treatment alone led to a weak and delayed activation of NF-κB and MAPK signaling without any effect on proinflammatory cytokine production. By contrast, Sa15-21 failed to induce the activation of interferon regulatory factor 3. Taken together, our results indicate that Sa15-21 sensitizes macrophages to facilitate the inflammatory response via TLR signaling.

Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice.

The extracellular matrix (ECM) is the basis for virtually all cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrins and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at the middle of the molecule. Blockade of B4-FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. The unexpected nature of the B4-FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

Receptor-destroying enzyme (RDE) from Vibrio cholerae modulates IgE activity and reduces the initiation of anaphylaxis.

IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)-treated IgE cannot access its binding site on bone marrow-derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)-treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies.

Bone marrow PDGFRα+Sca-1+-enriched mesenchymal stem cells support survival of and antibody production by plasma cells in vitro through IL-6.

Plasma cells (PCs) acquiring long lifespans in the bone marrow (BM) play a pivotal role in the humoral arm of immunological memory. The PCs reside in a special BM niche and produce antibodies against past-encountered pathogens or vaccine components for a long time. In BM, cysteine-X-cysteine (CXC) chemokine receptor type 4 (CXCR4)-expressing PCs and myeloid cells such as dendritic cells are attracted to and held by CXC chemokine ligand 12 (CXCR12)-secreting stromal cells, where survival of the PCs is supported by soluble factors such as IL-6 and APRIL (a proliferation-inducing ligand) produced by neighboring myeloid cells. Although these stromal cells are also supposed to be involved in the support of the survival and antibody production, the full molecular mechanism has not been clarified yet. Here, we show that BM PDGFRα+Sca-1+-enriched mesenchymal stem cells (MSCs), which can contribute as stromal cells for hematopoietic stem cells, also support in vitro survival of and antibody production by BM PCs. IL-6 produced by MSCs was found to be involved in the support. Immunohistochemistry of BM sections suggested a co-localization of a minor population of PCs with PDGFRα+Sca-1+ MSCs in the BM. We also found that the sort-purified MSC preparation was composed of multiple cell groups with different gene expression profiles, as found on single-cell RNA sequencing, to which multiple roles in the in vitro PC support could be attributed.

Endoplasmic Protein Nogo-B (RTN4-B) Interacts with GRAMD4 and Regulates TLR9-Mediated Innate Immune Responses.

TLRs are distributed in their characteristic cellular or subcellular compartments to efficiently recognize specific ligands and to initiate intracellular signaling. Whereas TLRs recognizing pathogen-associated lipids or proteins are localized to the cell surface, nucleic acid-sensing TLRs are expressed in endosomes and lysosomes. Several endoplasmic reticulum (ER)-resident proteins are known to regulate the trafficking of TLRs to the specific cellular compartments, thus playing important roles in the initiation of innate immune responses. In this study, we show that an ER-resident protein, Nogo-B (or RTN4-B), is necessary for immune responses triggered by nucleic acid-sensing TLRs, and that a newly identified Nogo-B-binding protein (glucosyltransferases, Rab-like GTPase activators and myotubularins [GRAM] domain containing 4 [GRAMD4]) negatively regulates the responses. Production of inflammatory cytokines in vitro by macrophages stimulated with CpG-B oligonucleotides or polyinosinic:polycytidylic acid was attenuated in the absence of Nogo-B, which was also confirmed in serum samples from Nogo-deficient mice injected with polyinosinic:polycytidylic acid. Although a deficiency of Nogo-B did not change the incorporation or delivery of CpG to endosomes, the localization of TLR9 to endolysosomes was found to be impaired. We identified GRAMD4 as a downmodulator for TLR9 response with a Nogo-B binding ability in ER, because our knockdown and overexpression experiments indicated that GRAMD4 suppresses the TLR9 response and knockdown of Gramd4 strongly enhanced the response in the absence of Nogo-B. Our findings indicate a critical role of Nogo-B and GRAMD4 in trafficking of TLR9.

Human CD43+ B cells are closely related not only to memory B cells phenotypically but also to plasmablasts developmentally in healthy individuals.

CD20(+)CD27(+)CD43(+) B (CD43(+) B) cells have been newly defined among PBMCs and proposed to be human B1 cells. However, it is controversial as to whether they are orthologs of murine B1 cells and how they are related to other B-cell populations, particularly CD20(+)CD27(+)CD43(-) memory B cells and CD20(low)CD27(high)CD43(high) plasmablasts. Our objective is to identify phenotypically the position of CD43(+) B cells among peripheral B-lineage cell compartments in healthy donors, with reference to B-cell subsets from patients with systemic lupus erythematosus (SLE). We found that CD43(+) B cells among PBMCs from healthy subjects were indistinguishable phenotypically from memory B cells in terms of surface markers, and spontaneous in vitro Ig and IL-10 secretion capability, but quite different from plasmablasts. However, a moderate correlation was found in the frequency of CD43(+) B cells with that of plasmablasts in healthy donors but not in SLE patients. An in vitro differentiation experiment indicated that CD43(+) B cells give rise to plasmablasts more efficiently than do memory B cells, suggesting that they are more closely related to plasmablasts developmentally than are memory B cells, which is also supported by quantitative PCR analysis of mRNA expression of B-cell and plasma cell signature genes. Thus, we conclude that, in healthy individuals, CD43(+) B cells are closely related not only to memory B cells phenotypically but also to plasmablasts developmentally, although the developmental origin of CD43(+) B cells is not necessarily the same as that of plasmablasts.

Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing.

BM-derived endothelial progenitor cells (EPCs) are critical and essential for neovascularization in tissue repair and tumorigenesis. EPCs migrate from BM to tissues via the bloodstream, but specific chemotactic cues have not been identified. Here we show in mice that the absence of CCR5 reduced vascular EPC accumulation and neovascularization, but not macrophage recruitment, and eventually delayed healing in wounded skin. When transferred into Ccr5-/- mice, Ccr5+/+ BM cells, but not Ccr5-/- cells, accumulated in the wound site, were incorporated into the vasculature, and restored normal neovascularization. Consistent with these observations, CCL5 induced in vitro EPC migration in a CCR5-dependent manner. Moreover, expression of VEGF and TGF-ß was substantially diminished at wound sites in Ccr5-/- mice, which suggests that EPCs are important not only as the progenitors of endothelial cells, but also as the source of growth factors during tissue repair. Taken together, these data identify the CCL5/CCR5 interaction as what we believe to be a novel molecular target for modulation of neovascularization and eventual tissue repair.

Protective roles of CX3CR1-mediated signals in toxin A-induced enteritis through the induction of heme oxygenase-1 expression.

The injection of Clostridium difficile toxin A into the ileal loops caused fluid accumulation with the destruction of intestinal epithelial structure and the recruitment of neutrophils and macrophages. Concomitantly, intraileal gene expression of CX3CL1/fractalkine (FKN) and its receptor, CX3CR1, was enhanced. When treated with toxin A in a similar manner, CX3CR1-deficient (CX3CR1(-/-)) mice exhibited exaggerated fluid accumulation, histopathological alterations, and neutrophil recruitment, but not macrophage infiltration. Mice reconstituted with CX3CR1(-/-) mouse-derived bone marrow cells exhibited exacerbated toxin A-induced enteritis, indicating that the lack of the CX3CR1 gene for hematopoietic cells aggravated toxin A-induced enteritis. A heme oxygenase-1 (HO-1) inhibitor, tin-protoporphyrin-IX, markedly increased fluid accumulation in toxin A-treated wild-type mice, indicating the protective roles of HO-1 in this situation. HO-1 expression was detected mainly in F4/80-positive cells expressing CX3CR1, and CX3CR1(-/-) mice failed to increase HO-1 expression after toxin A treatment. Moreover, CX3CL1/FKN induced HO-1 gene expression by isolated lamina propria-derived macrophages or a mouse macrophage cell line, RAW264.7, through the activation of the ERK signal pathway. Thus, CX3CL1/FKN could induce CX3CR1-expressing macrophages to express HO-1, thereby ameliorating toxin A-induced enteritis.

Inhibitory immunoglobulin-like receptors LILRB and PIR-B negatively regulate osteoclast development.

Osteoclasts, multinucleated cells of myeloid-monocytic origin, are responsible for bone resorption, which is crucial for maintenance of bone homeostasis in concert with bone-forming osteoblasts of nonhematopoietic, mesenchymal origin. Receptor activator of NF-kappaB ligand (RANKL) and M-CSF, expressed on the surface of and secreted by osteoblasts, respectively, are essential factors that facilitate osteoclast formation. In contrast to the activation processes for osteoclast formation, inhibitory mechanisms for it are poorly understood. Herein we demonstrate that inhibitory Ig-like receptors recruiting Src homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) are expressed on osteoclast precursor cells like other myeloid cells, and that they play a regulatory role in the development of osteoclasts. We detected cell-surface expression of paired Ig-like receptor (PIR)-B and four isoforms of leukocyte Ig-like receptor (LILR)B on cultured osteoclast precursor cells of mouse and human origin, respectively, and showed that all of these ITIM-harboring inhibitory receptors constitutively recruit SHP-1 in the presence of RANKL and M-CSF, and that some of them can suppress osteoclast development in vitro. Fluorescence energy transfer analyses have suggested that the constitutive binding of either murine PIR-B or its human ortholog LILRB1 to MHC class I molecules on the same cell surface comprises one of the mechanisms for developmental regulation. These results constitute the first evidence of the regulation of osteoclast formation by cell-surface, ITIM-harboring Ig-like receptors. Modulation of these regulatory receptors may be a novel way to control various skeletal system disorders and inflammatory arthritis.

Plexin-A1 and its interaction with DAP12 in immune responses and bone homeostasis.

Semaphorins and their receptors have diverse functions in axon guidance, organogenesis, vascularization and/or angiogenesis, oncogenesis and regulation of immune responses. The primary receptors for semaphorins are members of the plexin family. In particular, plexin-A1, together with ligand-binding neuropilins, transduces repulsive axon guidance signals for soluble class III semaphorins, whereas plexin-A1 has multiple functions in chick cardiogenesis as a receptor for the transmembrane semaphorin, Sema6D, independent of neuropilins. Additionally, plexin-A1 has been implicated in dendritic cell function in the immune system. However, the role of plexin-A1 in vivo, and the mechanisms underlying its pleiotropic functions, remain unclear. Here, we generated plexin-A1-deficient (plexin-A1(-/-)) mice and identified its important roles, not only in immune responses, but also in bone homeostasis. Furthermore, we show that plexin-A1 associates with the triggering receptor expressed on myeloid cells-2 (Trem-2), linking semaphorin-signalling to the immuno-receptor tyrosine-based activation motif (ITAM)-bearing adaptor protein, DAP12. These findings reveal an unexpected role for plexin-A1 and present a novel signalling mechanism for exerting the pleiotropic functions of semaphorins.

Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis.

Costimulatory signals are required for activation of immune cells, but it is not known whether they contribute to other biological systems. The development and homeostasis of the skeletal system depend on the balance between bone formation and resorption. Receptor activator of NF-kappaB ligand (RANKL) regulates the differentiation of bone-resorbing cells, osteoclasts, in the presence of macrophage-colony stimulating factor (M-CSF). But it remains unclear how RANKL activates the calcium signals that lead to induction of nuclear factor of activated T cells c1, a key transcription factor for osteoclastogenesis. Here we show that mice lacking immunoreceptor tyrosine-based activation motif (ITAM)-harbouring adaptors, Fc receptor common gamma subunit (FcRgamma) and DNAX-activating protein (DAP)12, exhibit severe osteopetrosis owing to impaired osteoclast differentiation. In osteoclast precursor cells, FcRgamma and DAP12 associate with multiple immunoreceptors and activate calcium signalling through phospholipase Cgamma. Thus, ITAM-dependent costimulatory signals activated by multiple immunoreceptors are essential for the maintenance of bone homeostasis. These results reveal that RANKL and M-CSF are not sufficient to activate the signals required for osteoclastogenesis.

Signaling via immunoglobulin Fc receptors induces oligodendrocyte precursor cell differentiation.

Dramatic changes in morphology and myelin protein expression take place during the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes. Fyn tyrosine kinase was reported to play a central role in the differentiation process. Molecules that could induce Fyn signaling have not been studied. Such molecules are promising therapeutic targets in demyelinating diseases. We provide evidence that the common gamma chain of immunoglobulin Fc receptors (FcRgamma) is expressed in OPCs and has a role in triggering Fyn signaling. FcRgamma cross-linking by immunoglobulin G on OPCs promotes the activation of Fyn signaling and induces rapid morphological differentiation with upregulation of myelin basic protein (MBP) expression levels. Mice deficient in FcRgamma are hypomyelinated, and a significant reduction in MBP content is evident. Our findings indicate that the FcRgamma-Fyn-MBP cascade is pivotal during the differentiation of OPCs into myelinating oligodendrocytes, revealing an unexpected involvement of immunological molecules.