Positive and negative regulation of the Fcγ receptor-stimulating activity of RNA-containing immune complexes by RNase.

The U1RNP complex, Ro/SSA, and La/SSB are major RNA-containing autoantigens. Immune complexes (ICs) composed of RNA-containing autoantigens and autoantibodies are suspected to be involved in the pathogenesis of some systemic autoimmune diseases. Therefore, RNase treatment, which degrades RNA in ICs, has been tested in clinical trials as a potential therapeutic agent. However, no studies to our knowledge have specifically evaluated the effect of RNase treatment on the Fcγ receptor-stimulating (FcγR-stimulating) activity of RNA-containing ICs. In this study, using a reporter system that specifically detects FcγR-stimulating capacity, we investigated the effect of RNase treatment on the FcγR-stimulating activity of RNA-containing ICs composed of autoantigens and autoantibodies from patients with systemic autoimmune diseases such as systemic lupus erythematosus. We found that RNase enhanced the FcγR-stimulating activity of Ro/SSA- and La/SSB-containing ICs, but attenuated that of the U1RNP complex-containing ICs. RNase decreased autoantibody binding to the U1RNP complex, but increased autoantibody binding to Ro/SSA and La/SSB. Our results suggest that RNase enhances FcγR activation by promoting the formation of ICs containing Ro/SSA or La/SSB. Our study provides insights into the pathophysiology of autoimmune diseases involving anti-Ro/SSA and anti-La/SSB autoantibodies, and into the therapeutic application of RNase treatment for systemic autoimmune diseases.

SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses.

Many patients with severe COVID-19 suffer from pneumonia and the elucidation of the mechanisms underlying the development of this severe condition is important. The in vivo function of the ORF8 protein secreted by SARS-CoV-2 is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2 and found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrated that the serum ORF8 protein levels are well-correlated with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a SARS-CoV-2 viral cytokine involved in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.

Abrogation of self-tolerance by misfolded self-antigens complexed with MHC class II molecules.

Specific MHC class II alleles are strongly associated with susceptibility to various autoimmune diseases. Although the primary function of MHC class II molecules is to present peptides to helper T cells, MHC class II molecules also function like a chaperone to transport misfolded intracellular proteins to the cell surface. In this study, we found that autoantibodies in patients with Graves' disease preferentially recognize thyroid-stimulating hormone receptor (TSHR) complexed with MHC class II molecules of Graves' disease risk alleles, suggesting that the aberrant TSHR transported by MHC class II molecules is the target of autoantibodies produced in Graves' disease. Mice injected with cells expressing mouse TSHR complexed with MHC class II molecules, but not TSHR alone, produced anti-TSHR autoantibodies. These findings suggested that aberrant self-antigens transported by MHC class II molecules exhibit antigenic properties that differ from normal self-antigens and abrogate self-tolerance, providing a novel mechanism for autoimmunity.

Anti-Double-Stranded DNA Antibodies Recognize DNA Presented on HLA Class II Molecules of Systemic Lupus Erythematosus Risk Alleles.

OBJECTIVE: Specific HLA class II alleles are associated with susceptibility to systemic lupus erythematosus (SLE). The role of HLA class II molecules in SLE pathogenesis remains unclear, although anti-DNA antibodies are specific to SLE and correlate with disease activity. We previously demonstrated that misfolded proteins bound to HLA class II molecules are specific targets for the autoantibodies produced in autoimmune diseases. This study was undertaken to validate our hypothesis that DNA binds to HLA class II molecules in a manner similar to that of misfolded proteins and that DNA bound to HLA class II molecules is involved in SLE pathogenesis. METHODS: We analyzed the binding of DNA to HLA class II molecules, as well as the response of cells expressing anti-DNA B cell receptors (BCRs) to cells expressing the DNA/HLA class II complex. RESULTS: Efficient binding of DNA to HLA class II molecules was observed in risk alleles of SLE, such as HLA-DRB1*15:01. The efficiency of DNA binding to each HLA-DR allele was positively associated with the risk of SLE conferred by the HLA-DR allele. In addition, reporter cells carrying anti-DNA BCRs were activated by cells expressing DNA/HLA class II complexes. CONCLUSION: These results provide evidence that DNA bound to HLA class II molecules is involved in SLE pathogenesis.

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

Plasmodium falciparum RIFIN is a novel ligand for inhibitory immune receptor LILRB2.

Plasmodium falciparum causes the most severe form of malaria. Acquired immunity against P. falciparum provides insufficient protection even after repeated infections. Therefore, P. falciparum parasites might exploit inhibitory receptors for immune evasion. P. falciparum RIFINs are products of a multigene family consisting of 150-200 genes. Previously, we demonstrated that some RIFINs downregulate the immune response through the leukocyte immunoglobulin-like receptor (LILR) family inhibitory receptor, LILRB1, and leukocyte-associated immunoglobulin-like receptor 1, LAIR1. In this study, we further analyzed the expression of inhibitory receptor ligands on P. falciparum-infected erythrocytes and found that P. falciparum-infected erythrocytes expressed ligands for another LILR family inhibitory receptor, LILRB2, that recognizes HLA class I molecules as a host ligand. Furthermore, we identified that a specific RIFIN was a ligand for LILRB2 by using a newly developed RIFIN expression library. In addition, the domain 3 of LILRB2 was involved in RIFIN binding, whereas the domains 1 and 2 of LILRB2 were involved in the binding to HLA class I molecules. These results suggest that inhibitory receptor LILRB2 is also targeted by RIFIN for immune evasion of P. falciparum similar to LILRB1 and LAIR1.

A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing ß cells. The response of autoreactive T cells to ß cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as ß cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

RAG-Mediated DNA Breaks Attenuate PU.1 Activity in Early B Cells through Activation of a SPIC-BCLAF1 Complex.

Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development.

Transport of cellular misfolded proteins to the cell surface by HLA-B27 free heavy chain.

HLA class I molecules play a central role in the immune system by presenting peptide antigens to cytotoxic T cells. Although most HLA class I molecules are associated with ß2-microglobulin, HLA class I heavy chain that is not associated with ß2-microglobulin is also expressed on certain cells. We recently found that cellular misfolded proteins are transported to the cell surface by HLA class II molecules via association with their peptide-binding grooves. Furthermore, misfolded self-antigens bound to autoimmune disease-susceptible HLA class II molecules are the targets for autoantibodies produced in certain autoimmune diseases. In the present study, we found that misfolded proteins were also transported to the cell surface by specific HLA class I molecules including HLA-B27, which is strongly associated with ankylosing spondylitis. In addition, the efficiency with which HLA class I molecules encoded by each allele transport misfolded proteins to the cell surface was significantly correlated with HLA class I free heavy chain expression on that surface. Moreover, misfolded proteins were coprecipitated with HLA class I free heavy chain but not with correctly folded HLA class I molecules. These findings reveal a novel function of HLA class I molecules to transport misfolded proteins to the cell surface, which might help us to understand the pathogenesis of HLA class I-associated diseases.

FcγRIIIA-mediated activation of NK cells by IgG heavy chain complexed with MHC class II molecules.

Natural killer (NK) cells are a major FcγRIIIA-expressing lymphocyte population that mediate antibody-dependent cellular cytotoxicity. Although NK cells are critical for immunity against viruses and tumors, they are also activated in the joints of patients with rheumatoid arthritis (RA) and may be involved in disease progression. We previously found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins, such as IgG heavy chain (IgGH), to the cell surface via association with their peptide-binding grooves. Furthermore, we found that IgGHs bound to HLA class II molecules encoded by RA susceptibility alleles are specific targets for rheumatoid factor, an auto-antibody involved in RA. Here, we report that IgGHs bound to HLA class II molecules preferentially stimulate FcγRIIIA-expressing but not FcγRI-expressing cells. A significant correlation was observed between the reactivity of FcγRIIIA-expressing cells to IgGH complexed with a specific HLA-DR allele and the odds ratio for HLA-DR allele's association with RA. Moreover, primary human NK cells expressing FcγRIIIA demonstrated IFN-γ production and cytotoxicity against cells expressing IgGH complexed with HLA class II molecules. Our findings suggest that IgGH complexed with HLA class II molecules are involved in the activation of FcγRIIIA-expressing NK cells observed within arthritic joints.

Invariant chain p41 mediates production of soluble MHC class II molecules.

Major histocompatibility complex class II (MHC II) molecules are mainly expressed on antigen presentation cells and play an important role in immune response. It has been reported that MHC II molecules are also detected in serum as a soluble form (sMHC II molecules), and they are considered to be involved in the maintenance of self-tolerance. However, the mechanism by which sMHC II molecules are produced remains unclear. Invariant chain (Ii), also called CD74, plays an important role in antigen presentation of MHC II molecules. In the present study, we analyzed the role of Ii on the production of sMHC II molecules. We found that the amount of sMHC II molecules in serum was decreased in Ii-deficient mice compared to wild-type mice. sMHC II molecules were secreted from cells transfected with MHC II molecules and Ii but not from cells transfected with MHC II molecules alone. Moreover, isoform p41 of Ii-transfected cells induced more sMHC II molecules compared to isoform p31-transfected cells. The molecular weight of sMHC II molecules from MHC II and Ii p41-transfected cells was approximately 60 kDa, indicating that sMHC II molecules are a single heterodimer of α and ß chains that is not associated with micro-vesicles. From the analysis of Ii-deletion mutants, we found that the luminal domain of Ii p41 is crucial for the production of sMHC II molecules. These results suggested that Ii has an important role in production of sMHC II molecules.

Heme ameliorates dextran sodium sulfate-induced colitis through providing intestinal macrophages with noninflammatory profiles.

The local environment is crucial for shaping the identities of tissue-resident macrophages (Mϕs). When hemorrhage occurs in damaged tissues, hemoglobin induces differentiation of anti-inflammatory Mϕs with reparative function. Mucosal bleeding is one of the pathological features of inflammatory bowel diseases. However, the heme-mediated mechanism modulating activation of intestinal innate immune cells remains poorly understood. Here, we show that heme regulates gut homeostasis through induction of Spi-C in intestinal CX3CR1high Mϕs. Intestinal CX3CR1high Mϕs highly expressed Spi-C in a heme-dependent manner, and myeloid lineage-specific Spic-deficient (Lyz2-cre; Spicflox/flox ) mice showed severe intestinal inflammation with an increased number of Th17 cells during dextran sodium sulfate-induced colitis. Spi-C down-regulated the expression of a subset of Toll-like receptor (TLR)-inducible genes in intestinal CX3CR1high Mϕs to prevent colitis. LPS-induced production of IL-6 and IL-1α, but not IL-10 and TNF-α, by large intestinal Mϕs from Lyz2-cre; Spicflox/flox mice was markedly enhanced. The interaction of Spi-C with IRF5 was linked to disruption of the IRF5-NF-κB p65 complex formation, thereby abrogating recruitment of IRF5 and NF-κB p65 to the Il6 and Il1a promoters. Collectively, these results demonstrate that heme-mediated Spi-C is a key molecule for the noninflammatory signature of intestinal Mϕs by suppressing the induction of a subset of TLR-inducible genes through binding to IRF5.

Corrigendum: Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

This corrects the article DOI: 10.1038/nature24994.

Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.

Myeloperoxidase/HLA Class II Complexes Recognized by Autoantibodies in Microscopic Polyangiitis.

OBJECTIVE: Autoantibodies against myeloperoxidase (MPO) that are expressed in neutrophils play an important role in the pathogenesis of microscopic polyangiitis (MPA). We recently observed that misfolded cellular proteins are transported to the cell surface by HLA class II molecules and are targeted by autoantibodies in patients with rheumatoid arthritis or antiphospholipid syndrome, suggesting that HLA class II molecules play an important role in autoantibody recognition. The aim of this study was to address the role of HLA class II molecules in the cell surface expression of MPO in patients with MPA. METHODS: The association of MPO with HLA-DR was analyzed using MPO and HLA-DR transfectants as well as neutrophils from healthy donors and patients with MPA. Autoantibody binding to the MPO/HLA-DR complex was analyzed by flow cytometry. The association of MPO with HLA-DR was assessed using the immunoprecipitation technique. The function of MPO-antineutrophil cytoplasmic antibody (ANCA) was assessed using a neutrophil-like cell line expressing HLA-DR and MPO. RESULTS: MPO protein was detected on the cell surface in the presence of HLA-DR, and the MPO/HLA-DR complex was recognized by MPO-ANCA. A competitive inhibition assay suggested that MPO associated with HLA-DR expresses cryptic autoantibody epitopes for MPO-ANCA. Autoantibody binding to the MPO/HLA-DR complex was correlated with disease susceptibility conferred by each HLA-DR allele, suggesting that the MPO/HLA-DR complex is involved in the pathogenicity of MPA. Indeed, MPO-HLA class II complexes were detected in neutrophils from a patient with MPA as well as in cytokine-stimulated neutrophils from healthy donors. Moreover, MPO-ANCA stimulated MPO/HLA-DR complex-expressing HL-60 cells. CONCLUSION: Our findings suggest that MPO complexed with HLA class II molecules is involved in the pathogenesis of MPA as a target for MPO-ANCA.

Monocyte infiltration into obese and fibrilized tissues is regulated by PILRα.

Paired immunoglobulin-like type 2 receptor α (PILRα) is an inhibitory receptor that is mainly expressed on myeloid cells, and negatively regulates neutrophil infiltration during inflammation. However, PILRα role on monocyte has not been described. Under both steady-state and inflammatory conditions, monocytes migrate into tissues and differentiate into macrophages. Macrophages in adipose and liver tissues play important roles in tissue homeostasis and pathogenesis of metabolic diseases. Here, we found that PILRα controls monocyte mobility through regulating integrin signaling and inhibiting CD99-CD99 binding. Moreover, we found that Pilra(-/-) mice developed obesity and hepatomegaly with fibrosis, and the numbers of macrophages in adipose and liver tissues are significantly increased in Pilra(-/-) mice. These data suggest that immune inhibitory receptor, PILRα, plays an important role in the prevention of obesity and liver fibrosis.

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.

Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection.

ß2-Glycoprotein I/HLA class II complexes are novel autoantigens in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy complications. ß2-glycoprotein I (ß2GPI) complexed with phospholipid is recognized as a major target for autoantibodies in APS; however, less than half the patients with clinical manifestations of APS possess autoantibodies against the complexes. Therefore, the range of autoantigens involved in APS remains unclear. Recently, we found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins to the cell surface via association with their peptide-binding grooves. Furthermore, immunoglobulin G heavy chain/HLA class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we demonstrate that intact ß2GPI, not peptide, forms a complex with HLA class II molecules. Strikingly, 100 (83.3%) of the 120 APS patients analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize ß2GPI/HLA class II complexes in the absence of phospholipids. In situ association between ß2GPI and HLA class II was observed in placental tissues of APS patients but not in healthy controls. Furthermore, autoantibodies against ß2GPI/HLA class II complexes mediated complement-dependent cytotoxicity against cells expressing the complexes. These data suggest that ß2GPI/HLA class II complexes are a target in APS that might be involved in the pathogenesis.

Negative regulation of DSS-induced experimental colitis by PILRα.

Inflammatory bowel disease is thought to be a complex multifactorial disease, in which an increased inflammatory response plays an important role. Paired immunoglobulin-like type 2 receptor α (PILRα), well conserved in almost all mammals, is an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs in the cytoplasmic domain. PILRα is mainly expressed on myeloid cells and plays an important role in the regulation of inflammation. In the present study, we investigated the function of PILRα in inflammatory bowel disease using PILRα-deficient mice. When mice were orally administered dextran sulfate sodium (DSS), colonic mucosal injury and inflammation were significantly exacerbated in DSS-treated PILRα-deficient mice compared with wild-type (WT) mice. Flow cytometric analysis revealed that neutrophil and macrophage cell numbers were higher in the colons of DSS-treated PILRα-deficient mice than in those of WT mice. Blockade of CXCR2 expressed on neutrophils using a CXCR2 inhibitor decreased the severity of colitis observed in PILRα-deficient mice. These results suggest that PILRα negatively regulates inflammatory colitis by regulating the infiltration of inflammatory cells such as neutrophils and macrophages.