HMGB1: LPS Delivery Vehicle for Caspase-11-Mediated Pyroptosis.

Recognition of cytoplasmic lipopolysaccharide (LPS) by caspase-11 leads to pyroptosis and secretion of inflammatory mediators. In this issue of Immunity, Deng et al. (2018) report that high-mobility group box 1 (HMGB1) secreted by hepatocytes delivers extracellular LPS into the cytoplasm and mediates pyroptosis.

NRF2 is a spatiotemporal metabolic hub essential for the polyfunctionality of Th2 cells.

Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.

Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing new small molecule drugs.

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways.

A unique population of neutrophils generated by air pollutant-induced lung damage exacerbates airway inflammation.

BACKGROUND: Diesel exhaust particles (DEPs) are the main component of traffic-related air pollution and have been implicated in the pathogenesis and exacerbation of asthma. However, the mechanism by which DEP exposure aggravates asthma symptoms remains unclear. OBJECTIVE: This study aimed to identify a key cellular player of air pollutant-induced asthma exacerbation and development. METHODS: We examined the distribution of innate immune cells in the murine models of asthma induced by house dust mite and DEP. Changes in immune cell profiles caused by DEP exposure were confirmed by flow cytometry and RNA-Seq analysis. The roles of sialic acid-binding, Ig-like lectin F (SiglecF)-positive neutrophils were further evaluated by adoptive transfer experiment and in vitro functional studies. RESULTS: DEP exposure induced a unique population of lung granulocytes that coexpressed Ly6G and SiglecF. These cells differed phenotypically, morphologically, functionally, and transcriptionally from other SiglecF-expressing cells in the lungs. Our findings with murine models suggest that intratracheal challenge with DEPs induces the local release of adenosine triphosphate, which is a damage-associated molecular pattern signal. Adenosine triphosphate promotes the expression of SiglecF on neutrophils, and these SiglecF+ neutrophils worsen type 2 and 3 airway inflammation by producing high levels of cysteinyl leukotrienes and neutrophil extracellular traps. We also found Siglec8- (which corresponds to murine SiglecF) expressing neutrophils, and we found it in patients with asthma-chronic obstructive pulmonary disease overlap. CONCLUSION: The SiglecF+ neutrophil is a novel and critical player in airway inflammation and targeting this population could reverse or ameliorate asthma.

Bone marrow CX3CR1+ mononuclear cells relay a systemic microbiota signal to control hematopoietic progenitors in mice.

The microbiota regulate hematopoiesis in the bone marrow (BM); however, the detailed mechanisms remain largely unknown. In this study, we explored how microbiota-derived molecules (MDMs) were transferred to the BM and sensed by the local immune cells to control hematopoiesis under steady-state conditions. We reveal that MDMs, including bacterial DNA (bDNA), reach the BM via systemic blood circulation and are captured by CX3CR1+ mononuclear cells (MNCs). CX3CR1+ MNCs sense MDMs via endolysosomal Toll-like receptors (TLRs) to produce inflammatory cytokines, which control the basal expansion of hematopoietic progenitors, but not hematopoietic stem cells, and their differentiation potential toward myeloid lineages. CX3CR1+ MNCs colocate with hematopoietic progenitors at the perivascular region, and the depletion of CX3CR1+ MNCs impedes bDNA influx into the BM. Moreover, the abrogation of TLR pathways in CX3CR1+ MNCs abolished the microbiota effect on hematopoiesis. These studies demonstrate that systemic MDMs control BM hematopoiesis by producing CX3CR1+ MNC-mediated cytokines in the steady-state.

Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9.

Toll-like receptors (TLRs) activate the innate immune system in response to pathogens. Here we show that TLR9 proteolytic cleavage is a prerequisite for TLR9 signaling. Inhibition of lysosomal proteolysis rendered TLR9 inactive. The carboxy-terminal fragment of TLR9 thus generated included a portion of the TLR9 ectodomain, as well as the transmembrane and cytoplasmic domains. This cleavage fragment bound to the TLR9 ligand CpG DNA and, when expressed in Tlr9(-/-) dendritic cells, restored CpG DNA-induced cytokine production. Although cathepsin L generated the requisite TLR9 cleavage products in a cell-free in vitro system, several proteases influenced TLR9 cleavage in intact cells. Lysosomal proteolysis thus contributes to innate immunity by facilitating specific cleavage of TLR9.

CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Correction to: Effect of training on ultrasonography (US) BI-RADS features for radiology residents: a multicenter study comparing performances after training.

The original version of this article, published on 07 January 2019, unfortunately contained a mistake.

Effect of training on ultrasonography (US) BI-RADS features for radiology residents: a multicenter study comparing performances after training.

OBJECTIVES: To evaluate the effect of training radiology residents on breast ultrasonography (US) according to the Breast Imaging Reporting And Data System (BI-RADS) and the factors that influence the training effect. METHODS: This multicenter, prospective study was approved by eight institutional review boards. From September 2013 to July 2014, 248 breast masses in 227 women were included for US image acquisition. Representative B-mode and video images of the breast masses were recorded, among which 54 cases were included in the education set and 66 in the test set. Sixty-one radiology residents scheduled for breast imaging training individually reviewed the test set, immediately before, 1 month after, and 6 months after training. Diagnostic performances and US descriptors of the residents were evaluated and compared against those of expert radiologists. RESULTS: Agreements between residents and experienced radiologists showed improvement after training, while agreements between post-training and post-6-month training descriptors did not show significant differences (all p > 0.05, respectively). Sensitivity, negative predictive value (NPV), and AUC were significantly improved for residents post-training and post-6-month training (all p < 0.05), while approximating the performances of expert radiologists except for AUC (0.836, 0.840, and 0.908, respectively, p < 0.05). Low levels of pre-training AUC, total number of breast US examinations, and the number of sessions per week that residents were involved in were factors influencing the improvement of AUC. CONCLUSION: Training using education material dedicated for breast US imaging effectively improved the diagnostic performances of radiology residents and agreements with experienced radiologists on US BI-RADS features. KEY POINTS: • Agreements on lesion descriptors between residents and experienced radiologists showed improvement after training, regardless of test point. • Sensitivity, NPV, and AUC were significantly improved for residents in post-training and post-6-month training (all p < 0.05). • Low levels of pre-training AUC, total number of breast US examinations, and the number of sessions per week that residents were involved in were factors influencing the improvement of AUC.

Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice.

BACKGROUND & AIMS: Obesity and metabolic syndrome have been associated with alterations to the intestinal microbiota. However, few studies examined the effects of obesity on the intestinal immune system. We investigated changes in subsets of intestinal CD4+ T-helper (TH) cells with obesity and the effects of gut-tropic TH17 cells in mice on a high-fat diet (HFD). METHODS: We isolated immune cells from small intestine and adipose tissue of C57BL/6 mice fed a normal chow diet or a HFD for 10 weeks and analyzed the cells by flow cytometry. Mice fed a vitamin A-deficient HFD were compared with mice fed a vitamin A-sufficient HFD. Obese RAG1-deficient mice were given injections of only regulatory T cells or a combination of regulatory T cells and TH17 cells (wild type or deficient in integrin ß7 subunit or interleukin 17 [IL17]). Mice were examined for weight gain, fat mass, fatty liver, glucose tolerance, and insulin resistance. Fecal samples were collected before and after T cell transfer and analyzed for microbiota composition by metagenomic DNA sequencing and quantitative polymerase chain reaction. RESULTS: Mice placed on a HFD became obese, which affected the distribution of small intestinal CD4+ TH cells. Intestinal tissues from obese mice had significant reductions in the proportion of TH17 cells but increased proportion of TH1 cells, compared with intestinal tissues from nonobese mice. Depletion of vitamin A in obese mice further reduced the proportion of TH17 cells in small intestine; this reduction correlated with more weight gain and worsening of glucose intolerance and insulin resistance. Adoptive transfer of in vitro-differentiated gut-tropic TH17 cells to obese mice reduced these metabolic defects, which required the integrin ß7 subunit and IL17. Delivery of TH17 cells to intestines of mice led to expansion of commensal microbes associated with leanness. CONCLUSIONS: In mice, intestinal TH17 cells contribute to development of a microbiota that maintains metabolic homeostasis, via IL17. Gut-homing TH17 cells might be used to reduce metabolic disorders in obese individuals.

Lipids Regulate Lck Protein Activity through Their Interactions with the Lck Src Homology 2 Domain.

Lymphocyte-specific protein-tyrosine kinase (Lck) plays an essential role in T cell receptor (TCR) signaling and T cell development, but its activation mechanism is not fully understood. To explore the possibility that plasma membrane (PM) lipids control TCR signaling activities of Lck, we measured the membrane binding properties of its regulatory Src homology 2 (SH2) and Src homology 3 domains. The Lck SH2 domain binds anionic PM lipids with high affinity but with low specificity. Electrostatic potential calculation, NMR analysis, and mutational studies identified the lipid-binding site of the Lck SH2 domain that includes surface-exposed basic, aromatic, and hydrophobic residues but not the phospho-Tyr binding pocket. Mutation of lipid binding residues greatly reduced the interaction of Lck with the ζ chain in the activated TCR signaling complex and its overall TCR signaling activities. These results suggest that PM lipids, including phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, modulate interaction of Lck with its binding partners in the TCR signaling complex and its TCR signaling activities in a spatiotemporally specific manner via its SH2 domain.

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5.

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.

PIKfyve, a class III lipid kinase, is required for TLR-induced type I IFN production via modulation of ATF3.

Type I IFN plays a key role in antiviral responses. It also has been shown that deregulation of type I IFN expression following abnormal activation of TLRs contributes to the pathogenesis of systemic lupus erythematosus. In this study, we find that PIKfyve, a class III lipid kinase, is required for endolysosomal TLR-induced expression of type I IFN in mouse and human cells. PIKfyve binds to phosphatidylinositol 3-phosphate and synthesizes phosphatidylinositol 3,5-bisphosphate, and plays a critical role in endolysosomal trafficking. However, PIKfyve modulates type I IFN production via mechanisms independent of receptor and ligand trafficking in endolysosomes. Instead, pharmacological or genetic inactivation of PIKfyve rapidly induces expression of the transcription repressor ATF3, which is necessary and sufficient for suppression of type I IFN expression by binding to its promoter and blocking its transcription. Thus, we have uncovered a novel phosphoinositide-mediated regulatory mechanism that controls TLR-mediated induction of type I IFN, which may provide a new therapeutic indication for the PIKfyve inhibitor.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

UNLABELLED: T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. IMPORTANCE: Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Cell-specific TLR9 trafficking in primary APCs of transgenic TLR9-GFP mice.

Recognition of nucleic acids by TLR9 requires its trafficking from the endoplasmic reticulum to endolysosomal compartments and its subsequent proteolytic processing. Both processes depend on interactions of TLR9 with the polytopic endoplasmic reticulum-resident protein UNC93B1. To examine the intracellular behavior of TLR9 in primary APCs, we generated transgenic mice expressing a TLR9-GFP fusion. The TLR9-GFP transgene is functional and is proteolytically processed in resting bone marrow-derived macrophages (BMDMs), dendritic cells, and B cells. Inhibition of cleavage impairs TLR9-dependent responses in all primary APCs analyzed. The kinetics of TLR9-GFP processing in BMDMs and B cells differs: in B cells, proteolysis occurs at a faster rate, consistent with an almost exclusive localization to endolysosomes at the resting state. In contrast to the joint requirement for cathepsins L and S for TLR9 cleavage in macrophages, TLR9-GFP cleavage depends on cathepsin L activity in B cells. As expected, in BMDMs and B cells from UNC93B1 (3d) mutant mice, cleavage of TLR9-GFP is essentially blocked, and the expression level of UNC93B1 appears tightly correlated with TLR9-GFP cleavage. We conclude that proteolysis is a universal requirement for TLR9 activation in the primary cell types tested, however the cathepsin requirement, rate of cleavage, and intracellular behavior of TLR9 varies. The observed differences in trafficking indicate the possibility of distinct modes of endosomal content sampling to facilitate initiation of TLR-driven responses in APCs.

Intestinal Lin- c-Kit+ NKp46- CD4- population strongly produces IL-22 upon IL-1ß stimulation.

Small intestinal innate lymphoid cells (ILCs) regulate intestinal epithelial cell homeostasis and help to prevent pathogenic bacterial infections by producing IL-22. In a global gene-expression analysis comparing small intestinal ILCs (Lin(-)c-Kit(+)Sca-1(-) cells) with non-ILCs (Lin(-)c-Kit(-)Sca-1(-) cells), we found that Lin(-)c-Kit(+)Sca-1(-) cells highly expressed the mRNAs for Il22, antimicrobial peptides, Csf2rb2 (Il3r), mast cell proteases, and Rorc. We then subdivided the Lin(-)c-Kit(+)Sca-1(-) cells into three groups--Lin(-)c-Kit(+)NKp46(-)CD4(-), Lin(-)c-Kit(+)NKp46(-)CD4(+) (CD4(+) LTi-like cells), and Lin(-)c-Kit(+)NKp46(+) (NKp46(+) ILC22 cells)--and showed that the Lin(-)c-Kit(+)NKp46(-)CD4(-) cells produced the highest level of IL-22 protein after IL-1ß, IL-23, or IL-1ß and IL-23 stimulation. In addition, we showed that the majority of the Lin(-)c-Kit(+)NKp46(-)CD4(-) population was IL-7Rα(+)CD34(-)ß7(int) cells, and IL-7Rα(-) cells could be divided into three subsets (CD34(+)ß7(int), CD34(-)ß7(int), and CD34(int)ß7(hi) cells). The IL-7Rα(+)CD34(-)ß7(int) cells strongly expressed the transcripts for Il17f and Il22 after costimulation with IL-1ß and IL-23. The IL-7Rα(-)CD34(+)ß7(int) and IL-7Rα(-)CD34(int)ß7(hi) cells predominantly expressed the transcripts for mast cell proteases and differentiated almost entirely into mast cells after 1 wk in culture medium supplemented with a cytokine mixture, whereas the IL-7Rα(-)CD34(-)ß7(int) cells highly expressed α-defensins and showed no differentiation. Taken together, these findings indicate that the IL-7Rα(-)CD34(+)ß7(int) and IL-7Rα(-)CD34(int)ß7(hi) populations are mast cell progenitors, and the IL-7Rα(+)CD34(-)ß7(int) (CD4(-) LTi-like cells) and IL-7Rα(-)CD34(-)ß7(int) populations within Lin(-)c-Kit(+)NKp46(-)CD4(-) cells may control intestinal homeostasis and provide intestinal protection by producing high levels of IL-22 and α-defensins, respectively.

Acidic amino acid residues in the juxtamembrane region of the nucleotide-sensing TLRs are important for UNC93B1 binding and signaling.

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid-sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles.

Activation of NLRP3 and AIM2 inflammasomes by Porphyromonas gingivalis infection.

Porphyromonas gingivalis, a major periodontopathogen, is involved in the pathogenesis of periodontitis. Interleukin-1ß (IL-1ß), a proinflammatory cytokine, regulates innate immune responses and is critical for the host defense against bacterial infection. However, excessive IL-1ß is linked to periodontal destruction. IL-1ß synthesis, maturation, and secretion are tightly regulated by Toll-like receptor (TLR) signaling and inflammasome activation. We found much higher levels of inflammasome components in the gingival tissues from patients with chronic periodontitis than in those from healthy controls. To investigate the molecular mechanisms by which P. gingivalis infection causes IL-1ß secretion, we examined the characteristics of P. gingivalis-induced signaling in differentiated THP-1 cells. We found that P. gingivalis induces IL-1ß secretion and inflammatory cell death via caspase-1 activation. We also found that P. gingivalis-induced IL-1ß secretion and pyroptic cell death required both NLRP3 and AIM2 inflammasome activation. The activation of the NLRP3 inflammasome was mediated by ATP release, the P2X7 receptor, and lysosomal damage. In addition, we found that the priming signal via TLR2 and TLR4 activation precedes P. gingivalis-induced IL-1ß release. Our study provides novel insight into the innate immune response against P. gingivalis infection which could potentially be used for the prevention and therapy of periodontitis.

UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes.

Signalling by means of toll-like receptors (TLRs) is essential for the development of innate and adaptive immune responses. UNC93B1, essential for signalling of TLR3, TLR7 and TLR9 in both humans and mice, physically interacts with these TLRs in the endoplasmic reticulum (ER). Here we show that the function of the polytopic membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors TLR7 and TLR9 from the ER to endolysosomes. In dendritic cells of 3d mice, which express an UNC93B1 missense mutant (H412R) incapable of TLR binding, neither TLR7 nor TLR9 exits the ER. Furthermore, the trafficking and signalling defects of the nucleotide-sensing TLRs in 3d dendritic cells are corrected by expression of wild-type UNC93B1. However, UNC93B1 is dispensable for ligand recognition and signal initiation by TLRs. To our knowledge, UNC93B1 is the first protein to be identified as a molecule specifically involved in trafficking of nucleotide-sensing TLRs. By inhibiting the interaction between UNC93B1 and TLRs it should be possible to achieve specific regulation of the nucleotide-sensing TLRs without compromising signalling via the cell-surface-disposed TLRs.