MicroRNAs of the miR-17∼92 family are critical regulators of T(FH) differentiation.

Follicular helper T cells (T(FH) cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell-specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised T(FH) differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell-specific expression of a transgene encoding miR-17∼92 spontaneously accumulated T(FH) cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4(+) T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in T(FH) differentiation and establish PHLPP2 as an important mediator of their function in this process.

Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes.

The pathogenic Listeria monocytogenes bacterium produces the flagellum as a locomotive organelle at or below 30°C outside the host, but it halts flagellar expression at 37°C inside the human host to evade the flagellum-induced immune response. Listeria monocytogenes GmaR is a thermosensor protein that coordinates flagellar expression by binding the master transcriptional repressor of flagellar genes (MogR) in a temperature-responsive manner. To understand the regulatory mechanism whereby GmaR exerts the antirepression activity on flagellar expression, we performed structural and mutational analyses of the GmaR-MogR system. At or below 30°C, GmaR exists as a functional monomer and forms a circularly enclosed multidomain structure via an interdomain interaction. GmaR in this conformation recognizes MogR using the C-terminal antirepressor domain in a unique dual binding mode and mediates the antirepressor function through direct competition and spatial restraint mechanisms. Surprisingly, at 37°C, GmaR rapidly forms autologous aggregates that are deficient in MogR neutralization capabilities.

Lactoferrin-derived chimeric peptide (LFch) strongly boosts TGFß1-mediated inducible Treg differentiation possibly through downregulating TCR/CD28 signalling.

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFß receptor III (TßRIII), and this activity was further enhanced by TGFß1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17-30] and lactoferrampin [aa 265-284]), along with TGFß1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFß1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFß1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFß1 receptor III (sTßRIII). Consistent with this, the activity of LFch on TGFß1-induced Foxp3 expression was also abrogated by treatment with sTßRIII. Finally, the TGFß1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFß1-induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

Lactoferrin Potentiates Inducible Regulatory T Cell Differentiation through TGF-ß Receptor III Binding and Activation of Membrane-Bound TGF-ß.

Lactoferrin (LF) is known to possess anti-inflammatory activity, although its mechanisms of action are not well-understood. The present study asked whether LF affects the commitment of inducible regulatory T cells (Tregs). LF substantially promoted Foxp3 expression by mouse activated CD4+T cells, and this activity was further enhanced by TGF-ß1. Interestingly, blocking TGF-ß with anti-TGF-ß Ab completely abolished LF-induced Foxp3 expression. However, no significant amount of soluble TGF-ß was released by LF-stimulated T cells, suggesting that membrane TGF-ß (mTGF-ß) is associated. Subsequently, it was found that LF binds to TGF-ß receptor III, which induces reactive oxygen species production and diminishes the expression of mTGF-ß-bound latency-associated peptide, leading to the activation of mTGF-ß. It was followed by phosphorylation of Smad3 and enhanced Foxp3 expression. These results suggest that LF induces Foxp3+ Tregs through TGF-ß receptor III/reactive oxygen species-mediated mTGF-ß activation, triggering canonical Smad3-dependent signaling. Finally, we found that the suppressive activity of LF-induced Tregs is facilitated mainly by CD39/CD73-induced adenosine generation and that this suppressor activity alleviates inflammatory bowel disease.

Frequency-Domain Reverse-Time Migration with Analytic Green's Function for the Seismic Imaging of Shallow Water Column Structures in the Arctic Ocean.

Seismic oceanography can provide a two- or three-dimensional view of the water column thermocline structure at a vertical and horizontal resolution from the multi-channel seismic dataset. Several seismic imaging methods and techniques for seismic oceanography have been presented in previous research. In this study, we suggest a new formulation of the frequency-domain reverse-time migration method for seismic oceanography based on the analytic Green's function. For imaging thermocline structures in the water column from the seismic data, our proposed seismic reverse-time migration method uses the analytic Green's function for numerically calculating the forward- and backward-modeled wavefield rather than the wave propagation modeling in the conventional algorithm. The frequency-domain reverse-time migration with analytic Green's function does not require significant computational memory, resources, or a multifrontal direct solver to calculate the migration seismic images as like conventional reverse-time migration. The analytic Green's function in our reverse-time method makes it possible to provide a high-resolution seismic water column image with a meter-scale grid size, consisting of full-band frequency components for a modest cost and in a low-memory environment for computation. Our method was applied to multi-channel seismic data acquired in the Arctic Ocean and successfully constructed water column seismic images containing the oceanographic reflections caused by thermocline structures of the water mass. From the numerical test, we note that the oceanographic reflections of the migrated seismic images reflected the distribution of Arctic waters in a shallow depth and showed good correspondence with the anomalies of measured temperatures and calculated reflection coefficients from each XCDT profile. Our proposed method has been verified for field data application and accuracy of imaging performance.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

Helicobacter pylori flagellin: TLR5 evasion and fusion-based conversion into a TLR5 agonist.

Helicobacter pylori is a flagellated bacterium of the Epsilonproteobacteria class that causes peptic ulcers. Flagellin is a primary structural protein that assembles into the flagellar filament. Flagellins from bacteria that belong to the Gammaproteobacteria and Firmicutes groups are detected by Toll-like receptor 5 (TLR5) in the host, triggering the innate immune response, and thus have been studied for the development of vaccines against diverse infections through fusion with protein antigens. However, H. pylori flagellin (hFlg) does not stimulate TLR5, allowing H. pylori to evade TLR5-mediated immune surveillance. The unresponsiveness of TLR5 to hFlg, along with the tendency of the hFlg protein to precipitate, limits the utility of hFlg for H. pylori vaccine development. Here, we report a soluble hFlg derivative protein that activates TLR5. We performed expression and purification screens with full-length and fragment hFlg proteins and identified the hypervariable domains as the soluble part of hFlg. The hypervariable domains of hFlg were engineered into a TLR5 agonist through fusion with the TLR5-activating Bacillus subtilis flagellin. Furthermore, based on comparative sequence and mutation analyses, we reveal that hFlg evolved to evade TLR5 detection by modifying residues that correspond to a TLR5-activation hot spot.

Wnt3A Induces GSK-3ß Phosphorylation and ß-Catenin Accumulation Through RhoA/ROCK.

In canonical pathway, Wnt3A has been known to stabilize ß-catenin through the dissociation between ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) that suppresses the phosphorylation and degradation of ß-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3ß and accumulation of ß-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3ß and accumulation of ß-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3ß phosphorylation and ß-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3ß in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3ß phosphorylation and ß-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

MicroRNA-17~92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways.

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR-17~92 expression is frequently found in human cancers, mainly due to gene amplification and Myc-mediated transcriptional upregulation. Here we show that B cell-specific miR-17~92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc-driven lymphomagenesis stringently requires two intact alleles of miR-17~92. We experimentally identified miR-17~92 target genes by PAR-CLIP and validated select target genes in miR-17~92 transgenic mice. These analyses demonstrate that miR-17~92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR-17~92-driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma-bearing mice. These findings establish miR-17~92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth.

Mechanism underlying the suppressor activity of retinoic acid on IL4-induced IgE synthesis and its physiological implication.

The present study extends an earlier report that retinoic acid (RA) down-regulates IgE Ab synthesis in vitro. Here, we show the suppressive activity of RA on IgE production in vivo and its underlying mechanisms. We found that RA down-regulated IgE class switching recombination (CSR) mainly through RA receptor α (RARα). Additionally, RA inhibited histone acetylation of germ-line ε (GL ε) promoter, leading to suppression of IgE CSR. Consistently, serum IgE levels were substantially elevated in vitamin A-deficient (VAD) mice and this was more dramatic in VAD-lecithin:retinol acyltransferase deficient (LRAT-/-) mice. Further, serum mouse mast cell protease-1 (mMCP-1) level was elevated while frequency of intestinal regulatory T cells (Tregs) were diminished in VAD LRAT-/- mice, reflecting that deprivation of RA leads to allergic immune response. Taken together, our results reveal that RA has an IgE-repressive activity in vivo, which may ameliorate IgE-mediated allergic disease.

Structural and biochemical characterization of the Bacillus cereus 3-hydroxyisobutyrate dehydrogenase.

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) family catalyzes the NAD(+)- or NADP(+)-dependent oxidation of various ß-hydroxyacid substrates into their cognate semialdehydes for diverse metabolic pathways. Because HIBADH group members exhibit different substrate specificities, the substrate-recognition mode of each enzyme should be individually characterized. In the current study, we report the biochemical and structural analysis of a HIBADH group enzyme from Bacillus cereus (bcHIBADH). bcHIBADH mediates a dehydrogenation reaction on S-3-hydroxyisobutyrate substrate with high catalytic efficiency in an NAD(+)-dependent manner; it also oxidizes l-serine and 3-hydroxypropionate with lower activity. bcHIBADH consists of two domains and is further assembled into a functional dimer rather than a tetramer that has been commonly observed in other prokaryotic HIBADH group members. In the bcHIBADH structure, the interdomain cleft forms a putative active site and simultaneously accommodates both an NAD(+) cofactor and a substrate mimic. Our structure-based comparative analysis highlights structural motifs that are important in the cofactor and substrate recognition of the HIBADH group.

Activation of p38α in T cells regulates the intestinal host defense against attaching and effacing bacterial infections.

Intestinal infections by attaching and effacing (A/E) bacterial pathogens cause severe colitis and bloody diarrhea. Although p38α in intestinal epithelial cells (IEC) plays an important role in promoting protection against A/E bacteria by regulating T cell recruitment, its impact on immune responses remains unclear. In this study, we show that activation of p38α in T cells is critical for the clearance of the A/E pathogen Citrobacter rodentium. Mice deficient of p38α in T cells, but not in macrophages or dendritic cells, were impaired in clearing C. rodentium. Expression of inflammatory cytokines such as IFN-γ by p38α-deficient T cells was reduced, which further reduced the expression of inflammatory cytokines, chemokines, and antimicrobial peptide by IECs and led to reduced infiltration of T cells into the infected colon. Administration of IFN-γ activated the mucosal immunity to C. rodentium infection by increasing the expression of inflammation genes and the recruitment of T cells to the site of infection. Thus, p38α contributes to host defense against A/E pathogen infection by regulating the expression of inflammatory cytokines that activate host defense pathways in IECs.

Combined Treatment With TGF-ß1, Retinoic Acid, and Lactoferrin Robustly Generate Inducible Tregs (iTregs) Against High Affinity Ligand.

Forkhead box P3-positive (Foxp3+)-inducible Tregs (iTregs) are readily generated by TGF-ß1 at low TCR signaling intensity. TGF-ß1-mediated Foxp3 expression is further enhanced by retinoic acid (RA) and lactoferrin (LF). However, the intensity of TCR signaling required for induction of Foxp3 expression by TGF-ß1 in combination with RA and LF is unknown. Here, we found that either RA or LF alone decreased TGF-ß1-mediated Foxp3 expression at low TCR signaling intensity. In contrast, at high TCR signaling intensity, the addition of either RA or LF strongly increased TGF-ß1-mediated Foxp3 expression. Moreover, decreased CD28 stimulation was more favorable for TGF-ß1/LF-mediated Foxp3 expression. Lastly, we found that at high signaling intensities of both TCR and CD28, combined treatment with TGF-ß1, RA, and LF induced robust expression of Foxp3, in parallel with powerful suppressive activity against responder T cell proliferation. Our findings that TGFß/RA/LF strongly generate high affinity Ag-specific iTreg population would be useful for the control of unwanted hypersensitive immune reactions such as various autoimmune diseases.

The miR-17∼92 miRNAs promote plasma cell differentiation by suppressing SOCS3-mediated NIK degradation.

The miR-17∼92 family microRNAs (miRNAs) play a key role in germinal center (GC) reaction through promoting T follicular helper (TFH) cell differentiation. It remains unclear whether they also have intrinsic functions in B cell differentiation and function. Here we show that mice with B cell-specific deletion of the miR-17∼92 family exhibit impaired GC reaction, plasma cell differentiation, and antibody production in response to protein antigen immunization and chronic viral infection. Employing CRISPR-mediated functional screening, we identify Socs3 as a key functional target of miR-17∼92 in regulating plasma cell differentiation. Mechanistically, SOCS3, whose expression is elevated in miR-17∼92 family-deficient B cells, interacts with NIK and promotes its ubiquitination and degradation, thereby impairing NF-κB signaling and plasma cell differentiation. This moderate increase in SOCS3 expression has little effect on IL-21-STAT3 signaling. Our study demonstrates differential sensitivity of two key signaling pathways to alterations in the protein level of an miRNA target gene.

Disruption of IL-18 signaling via engineered IL-18BP biologics alleviates experimental cholestatic liver disease.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis around intrahepatic and extrahepatic bile ducts leading to severe hepatic cirrhosis and high mortality. Although there is an urgent clinical unmet need for PSC, no effective medical therapy has been developed to delay the disease progression until today. IL-18 binding protein (IL-18BP) is well-known to be a natural negative feedback regulator for IL-18, and we have developed a recombinant long-acting IL-18BP referred to as APB-R3 as a therapeutic agent to treat IL-18-related inflammatory diseases. Here, we aimed to study whether disrupted IL-18 signaling by APB-R3 treatment can inhibit PSC injuries in the experimental DDC diet-induced PSC rodent model. First, we found that the amounts of free IL-18 are augmented under PSC condition with increased expression of biliary IL-18 receptors. Administration of APB-R3 effectively attenuated key diagnostic parameters of PSC such as plasma ALP and GGT levels as well as bile acids levels. We also observed that blockade of IL-18 suppressed ductular reactive and proliferative phenotypes of cholangiocytes. Additionally, APB-R3 significantly ameliorated DDC diet-induced periductal fibrosis and transcriptional expressions of pro-fibrotic marker genes. Enhanced senescence associated secretory phenotype (SASP) markers in cholestatic liver disease were diminished by APB-R3 treatment. Our findings clearly demonstrate that the administration of IL-18BP biologics, APB-R3, effectively alleviates DDC diet-induced biliary injuries in rodent PSC model, implying APB-R3 can be a promising therapeutic reagent which warrants clinical human trials as new therapeutic options.

Oral administration of Faecalibacterium prausnitzii and Akkermansia muciniphila strains from humans improves atopic dermatitis symptoms in DNCB induced NC/Nga mice.

Atopic dermatitis (AD) is a common inflammatory skin disease, and its pathogenesis is closely associated with microbial homeostasis in the gut, namely the gut-skin axis. Particularly, recent metagenomics studies revealed that the abundance of two major bacterial species in the gut, Faecalibacterium prausnitzii and Akkermansia muciniphila, may play a critical role in the pathogenesis of AD, but the effect of these species in AD has not yet been elucidated. To evaluate the potential beneficial effect of F. prausnitzii or A. muciniphila in AD, we conducted an animal model study where F. prausnitzii EB-FPDK11 or A. muciniphila EB-AMDK19, isolated from humans, was orally administered to 2,5-dinitrochlorobenzene (DNCB)-induced AD models using NC/Nga mice at a daily dose of 108 CFUs/mouse for six weeks. As a result, the administration of each strain of F. prausnitzii and A. muciniphila improved AD-related markers, such as dermatitis score, scratching behavior, and serum immunoglobulin E level. Also, the F. prausnitzii and A. muciniphila treatments decreased the level of thymic stromal lymphopoietin (TSLP), triggering the production of T helper (Th) 2 cytokines, and improved the imbalance between the Th1 and Th2 immune responses induced by DNCB. Meanwhile, the oral administration of the bacteria enhanced the production of filaggrin in the skin and ZO-1 in the gut barrier, leading to the recovery of functions. Taken together, our findings suggest that F. prausnitzii EB-FPDK11 and A. muciniphila EB-AMDK19 have a therapeutic potential in AD, which should be verified in humans.

Effects of Live and Pasteurized Forms of Akkermansia from the Human Gut on Obesity and Metabolic Dysregulation.

Akkermansia muciniphila (A. muciniphila) is a promising probiotic candidate owing to its health-promoting properties. A previous study reported that the pasteurized form of A. muciniphila strains isolated from human stool samples had a beneficial impact on high-fat diet-induced obese mice. On the other hand, the differences in the probiotic effects between live and pasteurized A. muciniphila on the metabolism and immune system of the host are still inconclusive. This study examines the differences between the live and pasteurized forms of A. muciniphila strains on the lipid and glucose metabolism and on regulating the inflammatory immune responses using a HFD-fed obese mouse model. The animals were administered the live and pasteurized forms of two A. muciniphila strains five times per week for the entire study period of 12 weeks. Both forms of the bacterial strains improved the HFD-induced obesity and metabolic dysregulation in the mice by preventing body-weight gains after one week. In addition, they cause a decrease in the weights of the major adipose tissues, adipogenesis/lipogenesis and serum TC levels, improvement in glucose homeostasis and suppression of inflammatory insults. Furthermore, these treatments restored the damaged gut architecture and integrity and improved the hepatic structure and function in HFD-induced animals. On the other hand, for both bacterial strains, the pasteurized form was more potent in improving glucose tolerance than the live form. Moreover, specific A. muciniphila preparations with either live or pasteurized bacteria decreased the number and population (%) of splenic Treg cells (CD4+ Foxp3+) significantly in the HFD-fed animals, further supporting the anti-inflammatory properties of these bacteria.

NLRP3 Triggers Attenuate Lipocalin-2 Expression Independent with Inflammasome Activation.

Lipocalin-2 (LCN2), a small secretory glycoprotein, is upregulated by toll-like receptor (TLR) signaling in various cells and tissues. LCN2 inhibits bacterial growth by iron sequestration and regulates the innate immune system. Inflammasome activates the inflammatory caspases leading to pyroptosis and cytokine maturation. This study examined the effects of inflammasome activation on LCN2 secretion in response to TLR signaling. The triggers of NLRP3 inflammasome activation attenuated LCN2 secretion while it induced interleukin-1ß in mouse macrophages. In mice, NLRP3 inflammasome activation inhibited TLR-mediated LCN2 secretion. The inhibition of NLRP3 triggers on LCN2 secretion was caused by the inhibited transcription and translation of LCN2. At the same time, no changes in the other cytokines and IκBζ, a well-known transcriptional factor of Lcn2 transcription, were observed. Overall, NLRP3 triggers are a regulator of LCN2 expression suggesting a new linkage of inflammasome activation and LCN2 secretion in the innate immunity.