Aberrant RNA sensing in regulatory T cells causes systemic autoimmunity.

Chronic and aberrant nucleic acid sensing causes type I IFN-driven autoimmune diseases, designated type I interferonopathies. We found a significant reduction of regulatory T cells (Tregs) in patients with type I interferonopathies caused by mutations in ADAR1 or IFIH1 (encoding MDA5). We analyzed the underlying mechanisms using murine models and found that Treg-specific deletion of Adar1 caused peripheral Treg loss and scurfy-like lethal autoimmune disorders. Similarly, knock-in mice with Treg-specific expression of an MDA5 gain-of-function mutant caused apoptosis of peripheral Tregs and severe autoimmunity. Moreover, the impact of ADAR1 deficiency on Tregs is multifaceted, involving both MDA5 and PKR sensing. Together, our results highlight the dysregulation of Treg homeostasis by intrinsic aberrant RNA sensing as a potential determinant for type I interferonopathies.

E3 ubiquitin ligases SIAH1/2 regulate hypoxia-inducible factor-1 (HIF-1)-mediated Th17 cell differentiation.

IL-17 is known to be a cytokine mainly secreted from Th17 cells, which well associate with autoimmune inflammatory responses. In the generation of Th17 cells, RORc and RORa have pivotal roles in controlling the transcription of Il17. We speculated additional regulation in Il17a transcription and randomly screened a 6344 clone cDNA library to identify specific modulators for Il17a promoter activity. After the screen, the E3 ubiquitin ligases SIAH1 and SIAH2 were investigated further and confirmed to increase Il17a promoter activity in a T-cell line and to promote Th17 development ex vivo. This enhancement was a consequence of enhanced expression of hypoxia-inducible factor-1α (HIF-1α) protein, which is reported to directly regulate expression of Il17a and Rorgt at the transcriptional level. In the absence of HIF-1α, both ubiquitin ligases had little effect on Th17 cell differentiation. These results suggest that the SIAH1 and SIAH2 play a pivotal role to promote Th17 cell differentiation through maintaining the stability of HIF-1α protein.

Neutrophil infiltration during inflammation is regulated by PILRα via modulation of integrin activation.

Acute inflammatory responses are important in host defense, whereas dysregulated inflammation results in life-threatening complications. Here we found that paired immunoglobulin-like type 2 receptor alpha (PILRα), an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), negatively regulated neutrophil infiltration during inflammation. Pilra(-/-) mice had increased neutrophil recruitment to inflammatory sites and were highly susceptible to endotoxin shock. Pilra(-/-) neutrophils showed enhanced transmigration ability and increased adhesion to the ß(2) integrin ligand ICAM-1. PILRα expressed on neutrophils constitutively associated in cis with its ligands, resulting in clustering of PILRα during stimulation with a chemoattractant. Clustering of PILRα enhanced ITIM-mediated signaling, thus modulating ß(2) integrin inside-out activation. These data demonstrate that neutrophil recruitment in inflammatory responses is regulated by PILRα via modulation of integrin activation.

PILRalpha is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein B.

Glycoprotein B (gB) is one of the essential components for infection by herpes simplex virus-1 (HSV-1). Although several cellular receptors that associate with glycoprotein D (gD), such as herpes virus entry mediator (HVEM) and Nectin-1, have been identified, specific molecules that mediate HSV-1 infection by associating with gB have not been elucidated. Here, we found that paired immunoglobulin-like type 2 receptor (PILR) alpha associates with gB, and cells transduced with PILRalpha become susceptible to HSV-1 infection. Furthermore, HSV-1 infection of human primary cells expressing both HVEM and PILRalpha was blocked by either anti-PILRalpha or anti-HVEM antibody. Our results demonstrate that cellular receptors for both gB and gD are required for HSV-1 infection and that PILRalpha plays an important role in HSV-1 infection as a coreceptor that associates with gB. These findings uncover a crucial aspect of the mechanism underlying HSV-1 infection.

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation.

6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of d-isoGln to l-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.

Role of toll-like receptors and their adaptors in adjuvant immunotherapy for cancer.

The potentiation of immune responses to tumor-associated antigen (Ag) is a pivotal issue in immunotherapy for cancer and thus requires the use of adjuvants, which are involved in efficient antibody (Ab) production and killer cell induction. The efficacy for tumor regression of a number of adjuvants that have been applied to immunotherapy in humans and tumor-bearing animal models has been tested without understanding of the function of adjuvants. Recent findings on the function of Toll-like receptors (TLRs) and their adaptors facilitated the elucidation of the molecular basis of adjuvant activity. TLR signaling was found to induce interferons (IFNs), chemokines and proinflammatory cytokines and mature dendritic cells (DCs) for enhanced efficiency in antigen presentation. The mediators then play a crucial role in the organization of acquired immunity and, together with matured DCs, activate cytotoxic T cells (CTL) and NK cells. These TLR outputs vary among adjuvants, which may depend on adjuvant-specific selection of appropriate sets of TLRs and their adaptors. Here we review how a variety of host immune responses are induced by an individual adjuvant to confer an adjuvant-specific anti-tumor immunity. We elaborate specifically on two adjuvants, BCG-cell wall skeleton and double-stranded RNA (dsRNA). The former activates TLR2/4 on DCs and induces tumor-specific CTL allowing general application to patients with surgically dissected cancer and improving prognosis, while the latter activates TLR3 on DCs to release type 1 IFN that induces tumor cell apoptosis and NK-mediated tumor cytotoxicity.

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan.

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.