The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in phagocytes.

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.

Anti-Interleukin-23 Autoantibodies in Adult-Onset Immunodeficiency.

BACKGROUND: Autoantibodies against interleukin-12 (anti-interleukin-12) are often identified in patients with thymoma, but opportunistic infections develop in only some of these patients. Interleukin-12 (with subunits p40 and p35) shares a common subunit with interleukin-23 (subunits p40 and p19). In a patient with disseminated Burkholderia gladioli infection, the identification of both anti-interleukin-23 and anti-interleukin-12 prompted further investigation. METHODS: Among the patients (most of whom had thymoma) who were known to have anti-interleukin-12, we screened for autoantibodies against interleukin-23 (anti-interleukin-23). To validate the potential role of anti-interleukin-23 with respect to opportunistic infection, we tested a second cohort of patients with thymoma as well as patients without either thymoma or known anti-interleukin-12 who had unusual infections. RESULTS: Among 30 patients with anti-interleukin-12 who had severe mycobacterial, bacterial, or fungal infections, 15 (50%) also had autoantibodies that neutralized interleukin-23. The potency of such neutralization was correlated with the severity of these infections. The neutralizing activity of anti-interleukin-12 alone was not associated with infection. In the validation cohort of 91 patients with thymoma, the presence of anti-interleukin-23 was associated with infection status in 74 patients (81%). Overall, neutralizing anti-interleukin-23 was detected in 30 of 116 patients (26%) with thymoma and in 30 of 36 patients (83%) with disseminated, cerebral, or pulmonary infections. Anti-interleukin-23 was present in 6 of 32 patients (19%) with severe intracellular infections and in 2 of 16 patients (12%) with unusual intracranial infections, including Cladophialophora bantiana and Mycobacterium avium complex. CONCLUSIONS: Among patients with a variety of mycobacterial, bacterial, or fungal infections, the presence of neutralizing anti-interleukin-23 was associated with severe, persistent opportunistic infections. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Immunometabolic Regulation of Bacterial Infection, Biofilms, and Antibiotic Susceptibility.

BACKGROUND: Upon infection, mucosal tissues activate a brisk inflammatory response to clear the pathogen, i.e., resistance to disease. Resistance to disease is orchestrated by tissue-resident macrophages, which undergo profound metabolic reprogramming after sensing the pathogen. These metabolically activated macrophages release many inflammatory factors, which promote their bactericidal function. However, in immunocompetent individuals, pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella evade this type of immunity, generating communities that thrive for the long term. SUMMARY: These organisms develop features that render them less susceptible to eradication, such as biofilms and increased tolerance to antibiotics. Furthermore, after antibiotic therapy withdrawal, "persister" cells rapidly upsurge, triggering inflammatory relapses that worsen host health. How these pathogens persisted in inflamed tissues replete with activated macrophages remains poorly understood. KEY MESSAGES: In this review, we discuss recent findings indicating that the ability of P. aeruginosa, S. aureus, and Salmonella to evolve biofilms and antibiotic tolerance is promoted by the similar metabolic routes that regulate macrophage metabolic reprogramming.

Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection.

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.

Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPß within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Their last will and testament: dying immune cells protect the urinary system with extracellular DNA traps.

Like most epithelial organs, the bladder and kidney can be directly accessed by bacteria evolved for invasion. Epithelia and immune cells attempt to stymie this infection with biophysical and chemical mechanisms. Goldspink et al. connected the Na+ gradient in the kidney medulla with an immune defense mounted by dead cells (namely, the explosive death of neutrophils and macrophages), resulting in extracellular DNA traps. The pathway from Na+ concentration to immune death is depicted.

Tongue sole creatine kinases function as DAMP and activate antimicrobial immunity via TLR2.

Creatine kinase (CK) is an enzyme that regulates adenosine triphosphate (ATP) metabolism to maintain energy homeostasis. Although CK has been reported to be involved in pathogen infection, the immune function of CK remains elusive. In this study, we identified two muscle-type CK from the teleost tongue sole Cynoglossus semilaevis (designated CsCKM-1 and CsCKM-2). Bacterial infection modulated CsCKM-1/2 expression in tongue sole tissues and induced the release of CsCKM-1/2 into serum. Recombinant CsCKM-1/2 (rCsCKM-1/2) exhibited robust kinase activity and bound to bacterial pathogens and pathogen-associated molecular patterns. rCsCKM-1/2 also bound to tongue sole peripheral blood leukocytes (PBLs) and promoted PBLs to uptake bacterial pathogens, inhibit bacterial proliferation, and express proinflammatory cytokines. When co-expressed in HEK293T cells, CsCKM-1/2 were found to interact with the leucine rich domain of toll-like receptor 2 (TLR2). The presence of TLR2 antagonist significantly reduced CsCKM-1/2-induced immune response and antibacterial effect. Taken together, these results indicated that tongue sole creatine kinases function as damage-associated molecular pattern (DAMP) molecules and play an important role in antimicrobial immunity via TLR2.

The dietary sweetener sucralose is a negative modulator of T cell-mediated responses.

Artificial sweeteners are used as calorie-free sugar substitutes in many food products and their consumption has increased substantially over the past years1. Although generally regarded as safe, some concerns have been raised about the long-term safety of the consumption of certain sweeteners2-5. In this study, we show that the intake of high doses of sucralose in mice results in immunomodulatory effects by limiting T cell proliferation and T cell differentiation. Mechanistically, sucralose affects the membrane order of T cells, accompanied by a reduced efficiency of T cell receptor signalling and intracellular calcium mobilization. Mice given sucralose show decreased CD8+ T cell antigen-specific responses in subcutaneous cancer models and bacterial infection models, and reduced T cell function in models of T cell-mediated autoimmunity. Overall, these findings suggest that a high intake of sucralose can dampen T cell-mediated responses, an effect that could be used in therapy to mitigate T cell-dependent autoimmune disorders.

An evolutionary trade-off between host immunity and metabolism drives fatty liver in male mice.

Adaptations to infectious and dietary pressures shape mammalian physiology and disease risk. How such adaptations affect sex-biased diseases remains insufficiently studied. In this study, we show that sex-dependent hepatic gene programs confer a robust (~300%) survival advantage for male mice during lethal bacterial infection. The transcription factor B cell lymphoma 6 (BCL6), which masculinizes hepatic gene expression at puberty, is essential for this advantage. However, protection by BCL6 protein comes at a cost during conditions of dietary excess, which result in overt fatty liver and glucose intolerance in males. Deleting hepatic BCL6 reverses these phenotypes but markedly lowers male survival during infection, thus establishing a sex-dependent trade-off between host defense and metabolic systems. Our findings offer strong evidence that some current sex-biased diseases are rooted in ancient evolutionary trade-offs between immunity and metabolism.

A genome-wide association study in a large community-based cohort identifies multiple loci associated with susceptibility to bacterial and viral infections.

There is limited data on host-specific genetic determinants of susceptibility to bacterial and viral infections. Genome-wide association studies using large population cohorts can be a first step towards identifying patients prone to infectious diseases and targets for new therapies. Genetic variants associated with clinically relevant entities of bacterial and viral infections (e.g., abdominal infections, respiratory infections, and sepsis) in 337,484 participants of the UK Biobank cohort were explored by genome-wide association analyses. Cases (n = 81,179) were identified based on ICD-10 diagnosis codes of hospital inpatient and death registries. Functional annotation was performed using gene expression (eQTL) data. Fifty-seven unique genome-wide significant loci were found, many of which are novel in the context of infectious diseases. Some of the detected genetic variants were previously reported associated with infectious, inflammatory, autoimmune, and malignant diseases or key components of the immune system (e.g., white blood cells, cytokines). Fine mapping of the HLA region revealed significant associations with HLA-DQA1, HLA-DRB1, and HLA-DRB4 locus alleles. PPP1R14A showed strong colocalization with abdominal infections and gene expression in sigmoid and transverse colon, suggesting causality. Shared significant loci across infections and non-infectious phenotypes in the UK Biobank cohort were found, suggesting associations for example between SNPs identified for abdominal infections and CRP, rheumatoid arthritis, and diabetes mellitus. We report multiple loci associated with bacterial and viral infections. A better understanding of the genetic determinants of bacterial and viral infections can be useful to identify patients at risk and in the development of new drugs.

The Roles of NOD-like Receptors in Innate Immunity in Otitis Media.

Acute otitis media (AOM) can persist or lead to various complications in individuals in which the innate immune system is impaired. In this context, impaired expression of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR), an intracellular pathogen-recognition receptor (PRR), is involved in the etiology of OM in humans and animals, affecting its development, severity, chronicity, recurrence, and associated complications. To assess this relationship, we reviewed literature reports relating NLR expression patterns with the pathophysiology and clinical features of OM in the larger context of impaired innate immunity. We summarized the results of published studies on the expression of NLRs in animals and humans in acute otitis media (AOM), otitis media with effusion (OME), chronic otitis media (COM) with cholesteatoma, and COM without cholesteatoma. NLRs were expressed mainly in association with bacterial infection in AOM, OME, COM with cholesteatoma, and COM without cholesteatoma. In addition, expression of NLRs was affected by the presence or absence of bacteria, fluid characteristics, disease recurrence, tissue type, and repeated surgery. Various factors of the innate immune system are involved in the pathogenesis of OM in the middle ear. NLRs are expressed in AOM, OME, COM with cholesteatoma, and COM without cholesteatoma. Impaired NLR expression induced the development, chronicity and recurrence of OM and exacerbated associated complications, indicating that NLRs have important roles in the pathogenesis of OM.

Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Blocking GARP-mediated activation of TGF-ß1 did not alter innate or adaptive immune responses to bacterial infection or protein immunization in mice.

Transmembrane protein GARP binds latent TGF-ß1 to form GARP:(latent)TGF-ß1 complexes on the surface of several cell types including Tregs, B-cells, and platelets. Upon stimulation, these cells release active TGF-ß1. Blocking TGF-ß1 activation by Tregs with anti-GARP:TGF-ß1 mAbs overcomes resistance to PD1/PD-L1 blockade and induces immune-mediated regressions of murine tumors, indicating that Treg-derived TGF-ß1 inhibits anti-tumor immunity. TGF-ß1 exerts a vast array of effects on immune responses. For example, it favors differentiation of TH17 cells and B-cell switch to IgA production, two important processes for mucosal immunity. Here, we sought to determine whether treatment with anti-GARP:TGF-ß1 mAbs would perturb immune responses to intestinal bacterial infection. We observed no aggravation of intestinal disease, no systemic dissemination, and no alteration of innate or adaptative immune responses upon oral gavage of C. rodentium in highly susceptible Il22r-/- mice treated with anti-GARP:TGF-ß1 mAbs. To examine the effects of GARP:TGF-ß1 blockade on Ig production, we compared B cell- and TH cell- responses to OVA or CTB protein immunization in mice carrying deletions of Garp in Tregs, B cells, or platelets. No alteration of adaptive immune responses to protein immunization was observed in the absence of GARP on any of these cells. Altogether, we show that antibody-mediated blockade of GARP:TGF-ß1 or genetic deletion of Garp in Tregs, B cells or platelets, do not alter innate or adaptive immune responses to intestinal bacterial infection or protein immunization in mice. Anti-GARP:TGF-ß1 mAbs, currently tested for cancer immunotherapy, may thus restore anti-tumor immunity without severely impairing other immune defenses. PRéCIS: Immunotherapy with GARP:TGF-ß1 mAbs may restore anti-tumor immunity without impairing immune or inflammatory responses required to maintain homeostasis or host defense against infection, notably at mucosal barriers.

Different response of Acipenser gueldenstaedtii CRP/SAP and SAA to bacterial challenge and chronic thermal stress sheds light on the innate immune system of sturgeons.

Sturgeons are chondrostean fish critically endangered due to anthropogenic loss and degradation of natural habitat and overfishing for meat and caviar production. Consequently, sturgeon aquaculture has extensively developed lately, being Russian sturgeon (Acipenser gueldenstaedtii) the second most important species reared for caviar production. However, Russian sturgeon aquaculture in subtropical countries, such as Uruguay, confronts difficulties because fish have to endure excessive summertime warm temperatures, which weaken their innate defences facilitating opportunistic infections. To address this problem, we look for identifying putative acute phase proteins (APPs), which might be robust serum biomarkers of both infection and chronic thermal stress, applied to monitoring Russian sturgeon health status in farms. We focused on the C-Reactive Protein/Serum Amyloid P (CRP/SAP) pentraxin since the pentraxin family includes well-known APPs, better characterised in mammals than fish. We identified A.gueldenstaedtii CRP/SAP (AgCRP/SAP), as a member of the universal CRP/SAP pentraxin sub-family, and studied AgCRP/SAP involvement in sturgeon response to bacterial challenge and chronic thermal stress, in comparison with A. gueldenstaedtii Serum Amyloid A (AgSAA), a previously described positive APP. Results showed that AgCRP/SAP is a constitutive serum component that remained constant upon Aeromonas hydrophila challenge and chronic thermal stress. Contrastingly, serum AgSAA was subjected to regulation by bacterial and thermal stress challenges, showing a 50-fold increase and 3-fold decline in serum levels, respectively. Overall, results highlight the potential value of AgSAA, but not of AgCRP/SAP, as a biomarker of bacterial infection and the need to continue searching for robust chronic thermal stress biomarkers in sturgeons.

Cytokine Expression of Lung Bacterial Infection in Newly Diagnosed Adult Hematological Malignancies.

Adult patients with hematological malignancies are frequently accompanied by bacterial infections in the lungs when they are first diagnosed. Sputum culture, procalcitonin (PCT), C-reactive protein (CRP), body temperature, and other routinely used assays are not always reliable. Cytokines are frequently abnormally produced in adult hematological malignancies associated with a lung infection, it is uncertain if cytokines can predict lung bacterial infections in individuals with hematological malignancies. Therefore, we reviewed 541 adult patients newly diagnosed with hematological malignancies, of which 254 patients had lung bacterial infections and 287 patients had no other clearly diagnosed infections. To explore the predictive value of cytokines for pulmonary bacterial infection in adult patients with hematological malignancies. Our results show that IL-4, IL-6, IL-8, IL-10, IL-12P70, IL-1ß, IL-2, IFN-γ, TNF-α, TNF-ß and IL-17A are in the lungs The expression level of bacterially infected individuals was higher than that of patients without any infections (P<0.05). Furthermore, we found that 88.89% (200/225) of patients with IL-6 ≥34.12 pg/ml had a bacterial infection in their lungs. With the level of IL-8 ≥16.35 pg/ml, 71.67% (210/293) of patients were infected. While 66.10% (193/292) of patients had lung bacterial infections with the level of IL-10 ≥5.62 pg/ml. When IL-6, IL-8, and IL-10 were both greater than or equal to their Cutoff-value, 98.52% (133/135) of patients had lung bacterial infection. Significantly better than PCT ≥0.11 ng/ml [63.83% (150/235)], body temperature ≥38.5°C [71.24% (62/87)], CRP ≥9.3 mg/L [53.59% (112/209)] the proportion of lung infection. In general. IL-6, IL-8 and IL-10 are abnormally elevated in patients with lung bacterial infections in adult hematological malignancies. Then, the abnormal increase of IL-6, IL-8 and IL-10 should pay close attention to the possible lung bacterial infection in patients.

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei.

Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC's diversity and multiple immune activities.

Role of Specialized Pro-Resolving Mediators in Modifying Host Defense and Decreasing Bacterial Virulence.

Bacterial infection activates the innate immune system as part of the host's defense against invading pathogens. Host response to bacterial pathogens includes leukocyte activation, inflammatory mediator release, phagocytosis, and killing of bacteria. An appropriate host response requires resolution. The resolution phase involves attenuation of neutrophil migration, neutrophil apoptosis, macrophage recruitment, increased phagocytosis, efferocytosis of apoptotic neutrophils, and tissue repair. Specialized Pro-resolving Mediators (SPMs) are bioactive fatty acids that were shown to be highly effective in promoting resolution of infectious inflammation and survival in several models of infection. In this review, we provide insight into the role of SPMs in active host defense mechanisms for bacterial clearance including a new mechanism of action in which an SPM acts directly to reduce bacterial virulence.

Oxalate Alters Cellular Bioenergetics, Redox Homeostasis, Antibacterial Response, and Immune Response in Macrophages.

Individuals with calcium oxalate (CaOx) kidney stones can have secondarily infected calculi which may play a role in the development of recurrent urinary tract infection (UTI). Uropathogenic Escherichia coli (UPEC) is the most common causative pathogen of UTIs. Macrophages play a critical role in host immune defense against bacterial infections. Our previous study demonstrated that oxalate, an important component of the most common type of kidney stone, impairs monocyte cellular bioenergetics and redox homeostasis. The objective of this study was to investigate whether oxalate compromises macrophage metabolism, redox status, anti-bacterial response, and immune response. Monocytes (THP-1, a human monocytic cell line) were exposed to sodium oxalate (soluble oxalate; 50 µM) for 48 hours prior to being differentiated into macrophages. Macrophages were subsequently exposed to calcium oxalate crystals (50 µM) for 48 hours followed by UPEC (MOI 1:2 or 1:5) for 2 hours. Peritoneal macrophages and bone marrow-derived macrophages (BMDM) from C57BL/6 mice were also exposed to oxalate. THP-1 macrophages treated with oxalate had decreased cellular bioenergetics, mitochondrial complex I and IV activity, and ATP levels compared to control cells. In addition, these cells had a significant increase in mitochondrial and total reactive oxygen species levels, mitochondrial gene expression, and pro-inflammatory cytokine (i.e. Interleukin-1ß, IL-1ß and Interleukin-6, IL-6) mRNA levels and secretion. In contrast, oxalate significantly decreased the mRNA levels and secretion of the anti-inflammatory cytokine, Interleukin-10 (IL-10). Further, oxalate increased the bacterial burden of primary macrophages. Our findings demonstrate that oxalate compromises macrophage metabolism, redox homeostasis, and cytokine signaling leading to a reduction in anti-bacterial response and increased infection. These data highlight a novel role of oxalate on macrophage function.

Identification and Functional Analysis of Cytokine-Like Protein CLEC-47 in Caenorhabditis elegans.

A variety of effector proteins contribute to host defense in Caenorhabditis elegans. However, beyond lytic enzymes and antimicrobial peptides and proteins, little is known about the exact function of these infection-related effectors. This study set out to identify pathogen-dependent cytokine-like molecules, focusing on C-type lectin domain-containing proteins (CLECs). In total, 38 CLECs that are differentially regulated in response to bacterial infections have been previously identified by microarray and transcriptome sequencing (RNA-seq) analyses in C. elegans. We successfully cloned 18 of these 38 CLECs and chose to focus on CLEC-47 because, among these 18 cloned CLECs, it was the smallest protein and was recombinantly expressed at the highest levels in prokaryotic cells examined by SDS-PAGE. Quantitative real-time PCR (qRT-PCR/qPCR) showed that the expression of clec-47 was induced by a variety of Gram-positive bacterial pathogens, including Enterococcus faecium, Staphylococcus aureus, and Cutibacterium acnes, but was suppressed by the Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. By expressing CLEC-47 in HEK 293 cells, we showed that CLEC-47 is released into the culture media, which the Golgi apparatus inhibitors (brefeldin A [BFA] and GolgiStop) could block. Purified recombinant CLEC-47 (maltose binding protein [MBP]-CLEC-47-His) did not display antimicrobial activity against ESKAPE pathogen isolates but bound directly to murine macrophage J774A.1 cells. Recombinant CLEC-47 attracted and recruited J774A.1 cells in a chemotaxis assay. In addition, qPCR studies and enzyme-linked immunosorbent assays (ELISAs) showed that CLEC-47 activates J774A.1 cells in a dose- and time-dependent manner to express the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, and Macrophage Inflammatory Protein 2 (MIP-2). Moreover, C. elegans, fed with CLEC-47-expressing Escherichia coli, demonstrated enhanced expression of several antimicrobial proteins (CNC-1, CNC-2, CPR-1, and CPR-2) as well as the detoxification protein MTL-1. These data suggest that CLEC-47 functions as a novel cytokine-like signaling molecule and exemplify how the study of infection-related effectors in C. elegans can help elucidate the evolution of immune responses. IMPORTANCE A variety of effector proteins contribute to host defense in the nematode Caenorhabditis elegans. However, little is known about the exact function of these infection-related effectors beyond lytic enzymes and antimicrobial peptides and proteins. This study set out to identify pathogen-dependent cytokine-like molecules, and we focus on the C-type lectin domain-containing proteins (CLECs). Our data suggest that CLEC-47 functions as a novel cytokine-like signaling molecule and exemplify how the study of infection-related effectors in nematodes can help elucidate the evolution of immune responses.

Impact of Posttranslational Modification in Pathogenesis of Rheumatoid Arthritis: Focusing on Citrullination, Carbamylation, and Acetylation.

Rheumatoid arthritis (RA) is caused by prolonged periodic interactions between genetic, environmental, and immunologic factors. Posttranslational modifications (PTMs) such as citrullination, carbamylation, and acetylation are correlated with the pathogenesis of RA. PTM and cell death mechanisms such as apoptosis, autophagy, NETosis, leukotoxic hypercitrullination (LTH), and necrosis are related to each other and induce autoantigenicity. Certain microbial infections, such as those caused by Porphyromonasgingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella copri, can induce autoantigens in RA. Anti-modified protein antibodies (AMPA) containing anti-citrullinated protein/peptide antibodies (ACPAs), anti-carbamylated protein (anti-CarP) antibodies, and anti-acetylated protein antibodies (AAPAs) play a role in pathogenesis as well as in prediction, diagnosis, and prognosis. Interestingly, smoking is correlated with both PTMs and AMPAs in the development of RA. However, there is lack of evidence that smoking induces the generation of AMPAs.