Molecular Immunology

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

IntroductionMalaria is still a pressing global health challenge, especially in sub-Saharan Africa, where behavioral factors such as alcohol consumption may exacerbate its impact. The present study is aimed at investigating the pathogenesis of alcohol-exacerbated malaria in Plasmodium berghei-infected an animal model (mice). MethodsMale mice were separated into four treatment groups: control, alcohol control, P. berghei and P. berghei plus acute alcohol treatment groups. Animals were infected with malaria through intraperitoneal injection of P. berghei and an acute dose of ethanol (20% v/v) was introduced 48 hours post-infection. Parasitaemia was monitored using the Giemsa-stained thin blood smears. Haematological parameters were assessed using automated blood analyser. Liver function was evaluated by measuring serum levels of AST and ALT and cytokine profiles (TNF-, INF-{gamma}, IL-6, IL-1{beta}) were quantified using ELISA kits. ResultsResults show that acute alcohol intake led to a significant increase in parasitaemia in the P. berghei group (p<0.01). Haematological analysis revealed a significant (p<0.001) reduction in RBC count, haemoglobin levels, haematocrit percentage, platelet count and others in the P. berghei plus acute alcohol group. Liver enzyme assays revealed an elevated AST and ALT levels (p<0.001) in the P. berghei group. Cytokine analysis revealed a significant (p<0.01) upregulation of pro-inflammatory cytokines (TNF- INF-{gamma}, IL-1{beta} and IL-6), due to acute alcohol. These results suggest that alcohol exacerbates malaria pathogenesis by increasing parasitaemia, promoting immune dysregulation and liver injury, mediated by a shift toward a pro-inflammatory cytokine profile.

IgA nephropathy (IgAN) is a common primary glomerulonephritis characterized by glomerular immune-complex deposits with (co)dominant IgA. These deposits are enriched for IgA1 glycoforms with some O-glycans deficient in galactose (Gd-IgA1). Circulating Gd-IgA1 is bound by IgG autoantibodies to form immune complexes, some of which deposit in glomeruli. Genomic and immunologic studies indicate involvement of pro-inflammatory signaling pathways in the production of Gd-IgA1 in IgAN. Genomic studies identified multiple genetic loci associated with IgAN and suggested a convergence on the NF-{kappa}B pathway, including RELA, the gene encoding the NF-{kappa}B subunit p65. However, the mechanisms by which NF-{kappa}B pathways may affect O-glycosylation in IgA1-producing cells are unknown. Using EBV-immortalized B cells derived from peripheral-blood mononuclear cells of IgAN patients and healthy controls that have constitutively activated NF-{kappa}B, we report that inhibition of NF-{kappa}B/p65 by a selective IKK{beta} inhibitor TPCA-1 reduced phosphorylation of NF-{kappa}B/p65 at S536 and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This was likely related to reduced expression of C1GALT1 gene that encodes the enzyme responsible for galactosylation of IgA1 O-glycans. Flow-cytometry imaging revealed changes in nuclear translocation and co-localization of the NF-{kappa}B/p65 with co-transcriptional factor SP1, a transcriptional activator of C1GALT1, suggesting that NF-{kappa}B pathway affects IgA1 O-glycosylation via SP1 transcriptional control of C1GALT1 expression. Furthermore, prolonged IKK{beta} inhibition altered B cell subpopulations, enhancing generation of cells with a plasmablast-like phenotype, characterized by high SSC MFI and CD138 expression. Together, these findings provide functional evidence for involvement of NF-{kappa}B/p65 and its transcriptional partners in IgA1 O-glycosylation. HighlightsO_LIIKK{beta} inhibition reduced C1GALT1 expression and thereby increased galactose-deficient IgA1 (Gd-IgA1) production in immortalized human B cells. C_LIO_LISP1+ subpopulations, a transcriptional activator of C1GALT1, declined after sustained NF-{kappa}B inhibition. C_LIO_LINF-{kappa}B inhibition shifted a subpopulation of B cells into a plasmablast-like phenotype. C_LIO_LIThis study links NF-{kappa}B signaling with the GWAS-identified RELA susceptibility locus and IgA1 O-glycosylation. C_LI

C. burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. The disease is either asymptomatic or manifests as a mild flu-like illness, but pneumonia or hepatitis might also occur. In most cases, the infection is self-limiting and the pathogen is cleared. In a small percentage of patients, the host immune system fails to eliminate the pathogen, potentially allowing the development of chronic Q fever months or even years after primary infection. The elimination of the bacteria, and thereby prevention of disease onset, would require an inflammatory response. Inflammasomes are multimeric protein complexes that induce a pro-inflammatory response to combat pathogens. Here we show that C. burnetii fails to induce a strong activation of the non-canonical inflammasome, independently of its type IVB secretion system. However, the pathogen is unable to prevent external activation of the non-canonical inflammasome, which subsequently results in a reduction of the bacterial burden. Importantly, the acylation pattern of lipid A was identified to be involved in avoiding the activation of the non-canonical inflammasome. C. burnetii harbors a tetra-acylated lipid A. Modification of the C. burnetii lipid A to penta-/hexa-acylation resulted in increased secretion of IL1{beta} and reduced bacterial load. Together, these results suggest that the acylation pattern of lipid A constitutes an important immune evasion strategy of C. burnetii by failing to activate the non-canonical inflammasome. In addition, evidence was provided that oxygen limitation arrests activation of the NLRP3 inflammasome in murine BMDM, which might prevent efficient elimination of bacteria under hypoxic conditions, such as in granulomas or in inflamed tissue. AUTHOR SUMMARYSeveral pathogens have evolved mechanisms to persist in the human host, which allows reoccurring or late onset of infection. The human innate immune system has therefore established several pathways, including the inflammasome, to prevent bacterial survival. Here we show that the obligate intracellular pathogen Coxiella burnetii, the causative agent of Q fever, prevents detection by the non-canonical NLRP3 inflammasome. This is mediated by the acylation pattern of its lipid A. Altering this acylation pattern allows activation of the inflammasome and, consequently, improved clearance of the pathogen. This information opens new avenues to target the immune response to C. burnetii infection with the goal to eliminate the bacteria and thereby prevent disease.

BackgroundRising antimicrobial resistance rates, require new therapeutic approaches such as antimicrobial peptides (AMPs), which are part of the innate immune defense, as alternatives to antibiotics. In this study, we aim to unravel the antibacterial activity of human histone H1.2 peptide against Pseudomonas aeruginosa and its potential immune modulatory role. MethodsWe used a hemofiltrate peptide database for antimicrobial peptide prediction to identify novel human AMPs. Thirteen sequences of histone H1 were identified as putative AMPs, synthesized, and tested against bacterial ESKAPE pathogens in a radial diffusion assay. SYTOX green assay, electrophoretic mobility shift assay, and differential proteomics assays were conducted to determine the mode of action of H1.2 peptide fragment. A crystal violet assay was performed to evaluate the inhibition of biofilm formation. The cytotoxicity of the peptide was tested in LDH and Alamar assays. Finally, to visualize the contributions of H1.2 in NETs formation, scanning electron microscopy was performed. ResultsThe H1.2 peptide inhibited the growth of P. aeruginosa in a dose and pH-dependent manner without cytotoxicity towards mammalian THP-1 cells. It acts on intracellular targets to inhibit the growth of P. aeruginosa. STRING analysis from the differential proteomics assay showed that H1.2 targets the downregulation of proteins involved in the biogenesis of outer membrane proteins, including the folding and trafficking of outer membrane proteins across the cytoplasmic membrane. Scanning electron microscopy images showed that H1.2 forms NET-like structures capable of trapping and immobilizing P. aeruginosa. ConclusionThe characterized antimicrobial activity of H1.2 points to a role for human histone H1 fragments in innate immunity and may represent a promising approach for the development of novel antibacterial therapies. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/724237v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1778ddborg.highwire.dtl.DTLVardef@26430org.highwire.dtl.DTLVardef@ffbfa2org.highwire.dtl.DTLVardef@7e38ae_HPS_FORMAT_FIGEXP M_FIG C_FIG Sec transport and BAM complex system including chaperone proteins and quality control proteases are inhibited by H1.2 in Pseudomonas aeruginosa.Outer membrane proteins (OMPs) are synthesized in the cytoplasm and transported across the inner membrane via the Sec translocase, assisted by SecA/SecB or ribosomes. In the periplasm, they are escorted by chaperones such as SurA to the BAM complex for insertion into the outer membrane. Here, we show that H1.2, an antimicrobial peptide, targets membrane biogenesis in P. aeruginosa through downregulating Sec translocase (SecA/SecB and SecYEG), SurA, and BAM complex. Therefore, leading to improper transfer, folding and insertion of OMPs into the outer membrane. Normally, misfolded proteins are degraded by the protease MucD to prevent toxic aggregation in the bacteria. However, with H1.2 inhibiting MucD the proteotoxic stress is exacerbated, ultimately compromising bacterial homeostasis and viability. Figure created using BioRender.com.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Cholesterol immunometabolism is a critical controller of immunopathology in respiratory infections such as tuberculosis. Smith-Lemli-Opitz syndrome (SLOS) patients are affected by a loss of 7-dehydrocholesterol reductase (DHCR7) function and have elevated 7-dehydrocholesterol (7DHC) and reduced cholesterol. Increased 7DHC has been found to be protective against viral infections in a range of infection models however SLOS patients have a higher susceptibility to respiratory infection. Here we use the zebrafish-Mycobacterium marinum infection model to demonstrate a compromised innate immune response to bacterial infection in the absence of dhcr7. We correlate increased 7DHC with increased activation of the IRF3/type I interferon axis and demonstrate Irf3 is a targetable signaling node to restore anti-bacterial immunity in a dhcr7-depleted background. Plain English summaryLoss of 7-dehydrocholesterol reductase causes Smith-Lemli-Opitz syndrome. One of the metabolic features of Smith-Lemli-Opitz syndrome is increased 7-dehydrocholesterol (7DHC). We find increased 7DHC inhibits the ability of zebrafish to control mycobacterial infection by mis-activating an antiviral immune response at the expense of a protective anti-bacterial immune response. Our study suggests the susceptibility to respiratory infections and increased neuroinflammation in Smith-Lemli-Opitz syndrome could be treated by targeting the antiviral protein IRF3.

The mucosal chemoattractant C-X-C motif chemokine ligand 17 (CXCL17) was recently identified as a ligand for the orphan G protein-coupled receptor 25 (GPR25). Although CXCL17 orthologs have been identified in fishes, amphibians, and mammals, their presence in reptiles and birds remains unclear. In this study, we employed bioinformatic searches based on gene synteny and sequence features to identify CXCL17 orthologs in public databases. We identified functional CXCL17 orthologs in 46 reptilian species, including lizards, snakes, turtles, and alligators. In contrast, we found only non-functional gene relics in 22 bird species, suggesting the avian lineage lost functional CXCL17 during evolution. A recombinant reptilian CXCL17 from the loggerhead turtle (Caretta caretta), termed Cc-CXCL17, directly bound to and efficiently activated its corresponding receptor, Cc-GPR25, in a C-terminal fragment-dependent manner. Activation of Cc-GPR25 by Cc-CXCL17 also induced chemotactic movement of transfected human embryonic kidney (HEK) 293T cells. In cross-species activity assays, CXCL17s from human and tropical clawed frog could activate Cc-GPR25 albeit with lower potency, but fish orthologs lacked this activity; all tested CXCL17s had no detectable activity towards chicken GPR25, but Cc-CXCL17 had low activity towards mallard GPR25. Our findings demonstrate the presence of functional CXCL17 orthologs in extant reptiles and provide evidence for their evolutionary loss in birds, offering new insights into the phylogenetic distribution of the newly identified CXCL17-GPR25 signaling system.

BackgroundCytokines are immunomodulatory proteins that play central roles in regulating immune responses and represent attractive targets for cancer therapy. However, as single agents, cytokines have shown limited clinical benefit due to systemic toxicities and a short in vivo half-life. Our group has focused on engineering fusion cytokines (fusokines) that couple two cytokines into a single biologic to reprogram immune cell responses by enforcing non-canonical receptor engagement and signaling. A chimeric IL-6/IL-1{beta} fusokine was engineered to test the hypothesis that enforced co-engagement of IL-6 and IL-1{beta} signaling pathways would confer a gain-of-function phenotype in T cells and promote robust anti-tumor immunity. Here, we describe the immunomodulatory properties of IL6/1 fusokine and a method to deliver this fusokine to produce inhibition of ovarian tumor growth in a pre-clinical mouse model. MethodsLentiviral vectors encoding murine or human IL6/1 were designed using Vector Builder and expressed in either HEK293, CHO or ID8-F3 (p53-/-) cells depending on the downstream experiment to be conducted. IL6/1 expression was validated by ELISA and flow cytometry. Effects of human IL6/1 (hIL6/1) on T cell function (proliferation, memory phenotype, activation induced apoptosis) were monitored by flow cytometry. For in vivo studies, ID8-F3 murine ovarian cancer cells expressing mouse IL6/1 (mIL6/1) were administered intraperitoneally (I.P.) as a cell-based therapy to C57BL/6 female mice bearing established ID8-F3 luciferase tumors. Tumor progression was monitored by bioluminescence (BLI) imaging, and overall survival was evaluated. ResultshIL6/1 significantly enhanced T cell survival and selectively promoted activation and expansion of CD45RO memory T cells. mIL6/1 expressing ID8-F3 cells (ID8IL6/1) demonstrated stable transduction and sustained cytokine secretion. In vivo, ID8IL6/1 cell therapy significantly reduced tumor growth and improved overall survival compared to control groups, with 2 of 8 mice achieving complete tumor clearance. ConclusionThese findings indicate that IL6/1 fusokine enhances T cell survival and proliferation while promoting memory responses. Engineered cancer cells (ID8-F3) expressing mIL6/1 fusokine induced a strong anti-tumor response when delivered as a therapeutic vaccine in ovarian cancer mouse model. What is already known on this topicO_LIFusokines are a class of bifunctional proteins designed to achieve synergistic immune modulation. Previous studies in our lab have shown fusokine exhibit gain-of-function immunomodulating activity. Individually, IL-6 and IL-1{beta} are recognized for their roles in promoting T-cell proliferation and effector function. However, the potential for a fused IL-6/1 fusokine to reprogram the immune system and elicit a superior anti-tumor response in vivo in ovarian cancer model is not yet studied. C_LI What this study addsO_LIThis study develops a novel fusion cytokine (fusokine), combining IL-6 and IL-1{beta}, and demonstrate robust activation of T cells. In a preclinical ovarian cancer model, engineered cancer cells expressing IL6/1 used as a therapeutic vaccine showed significant tumor reduction and improved overall survival. C_LI How this study might affect research, practice or policyO_LIThis study demonstrates that in comparison to individual cytokines, fusokines have greater potential to activate T cell function and when delivered as a cell therapy, achieve clear therapeutic efficacy in an ovarian cancer model. Further translational and clinical studies may enable the development of novel and more effective fusokine cell therapy approaches for patients with ovarian cancer. C_LI

AimsPercutaneous coronary intervention has improved survival following myocardial infarction, yet strategies to further reduce infarct size are limited. This study investigates the role of cytotoxic {gamma}{delta}-T cells in ischemic cardiomyocyte death and potential therapeutic interventions to reduce infarct size. MethodsGenetic and pharmacological approaches were used to delete {gamma}{delta}-T cells and their specific proteins to assess their involvement in cardiomyocyte death using mouse models of permanent ligation (PL) and ischemia/reperfusion (IR). Results{gamma}{delta}-T cells accumulated in infarct zones within 6h post-PL, expressing IFN-{gamma}, TNF-, granzyme B, and perforin. Their deletion reduced infarct size by 73% (PL) and 64% (IR). They induced cardiomyocyte death via apoptosis, gasdermin E-dependent pyroptosis, and MLKL-dependent necroptosis; {gamma}{delta}-T cell depletion reduced apoptosis by 80% and pyroptosis by 38%, with perforin deletion yielding similar effects. Necroptosis, attributed to combined IFN-{gamma}/TNF- cytotoxicity, decreased by 67%. Cytoplasmic DNA (cDNA) in stressed cardiomyocytes activated the cGAS/STING pathway, inducing expression of chemoattractant MCP-1 and death signal RAE-1. These signals recruited and activated {gamma}{delta}-T cells, which then triggered the death of the stressed cardiomyocytes. STING inhibition suppressed these expressions, reducing {gamma}{delta}-T cell accumulation and infarct size. NKG2D-deficient {gamma}{delta}-T cells prevented activation and reduced infarct size. Administration of an anti-IFNAR antibody at PL onset markedly reduced infarct size. ConclusionEarly activation of cytotoxic {gamma}{delta}-T cells via cardiomyocyte stress signals contributes significantly to immunogenic cardiomyocyte death. Targeting the STING pathway and type I interferon signalling presents a promising therapeutic avenue to mitigate infarct size and improve outcomes.

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, remains a significant public health and veterinary problem in endemic regions. Although chemotherapy and control programs exist, the development of complementary immunotherapeutic tools is increasingly needed. This study evaluated the generation and functional activity of hyperimmune serum (HIS) produced in three adult male castrated llamas (Lama glama) immunized with antigenic material derived from protoscoleces (PSCs) of the parasite. Sera collected after each of the first six immunizations were assessed by ELISA to quantify antigen-specific IgG responses, and their biological effects were tested in vitro using viable PSCs. Motility was measured using video-assisted paired-image scoring across serial serum dilutions (1:2-1:2048), and the methylene blue exclusion assay was used to assess viability. Hyperimmune serum produced a clear, reproducible, dose-dependent inhibition of PSC motility and viability. Higher titers of early inoculations reduced motility by 70-85%, while sera from the fifth and sixth inoculations achieved complete suppression. Naive serum and PBS controls showed no inhibitory effect. ELISA titers strongly correlated with biological activity, indicating that higher humoral responses predicted functional inhibition. These findings demonstrate the feasibility of generating potent anti-Echinococcus granulosus polyclonal antibodies in camelids and support their potential application in passive immunization strategies. The study establishes a foundation for future development of llama-derived immunobiological reagents, including nanobody-based tools, for the control of cystic echinococcosis.

Systemic lupus erythematosus (SLE) is associated with infection susceptibility and altered innate immune function. Monocyte metabolism is linked to appropriate cytokine release and bacterial containment. We investigated cytokine production and metabolic programming in the monocyte population from SLE patients and healthy controls following lipopolysaccharide (LPS) stimulation. SLE monocytes displayed increased IL-10, TNF, and IL-8 production, with impaired IL-1{beta} induction. Metabolic profiling revealed altered substrate use, with increased glucose dependence and reduced fatty acid and amino acid oxidation after LPS stimulation. SLE patients exhibited reduced numbers of classical monocytes, expansion of intermediate monocytes, and dysregulated subset-specific metabolic reprogramming in response to LPS. This descriptive study provides a cornerstone for (i) understanding infection susceptibility in SLE, (ii) subset-resolved immunometabolic profiling as a tool in autoimmunity, and (iii) developing future metabolic-targeted therapeutic strategies HighlightsO_LIDescriptive mapping shows SLE monocytes are proinflammatory with glucose dependence after LPS C_LIO_LIClassical and intermediate SLE subsets show divergent baseline metabolic preferences versus healthy C_LIO_LISLE subsets display aberrant LPS responses, i.e.. increased glucose and reduced fatty acid oxidation C_LIO_LIThis study provides a cornerstone for subset-resolved immunometabolism in infection susceptibility. C_LI

Osteolytic bone diseases are driven by excessive osteoclast formation and bone resorption. While cGAS-STING signaling is known to regulate bone homeostasis via macrophage-intrinsic mechanisms, its role in osteoblast-mediated control of osteoclastogenesis remains poorly defined. Here, we show that cGAS-STING activation of macrophages suppresses their osteoclastogenic potential while promoting immune activation. In osteoblasts, cGAS-STING triggers IRF3-mediated IFN-{beta} production and, notably, shifts the OPG-RANKL axis toward increased osteoprotegerin. In transwell co-culture, pre-activated osteoblasts reduce osteoclast differentiation of strain-matched macrophages. Mechanistically, osteoblast-derived IFN-{beta} is sufficient to inhibit osteoclastogenesis in a paracrine manner. Furthermore, autocrine IFN-{beta} signaling appears to modulate the OPG-RANKL axis, although additional regulatory factors may contribute. Together, these findings identify cGAS-STING-IFN-{beta} signaling as a dual regulator of osteoclastogenesis, acting directly on macrophages and indirectly via osteoblast-derived anti-osteoclastogenic mediators. This highlights osteoblasts as cGAS-STING-responsive bystander cells within the bone microenvironment that can be targeted as an alternative strategy to limit pathological bone resorption. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/724040v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@167dfcorg.highwire.dtl.DTLVardef@a95477org.highwire.dtl.DTLVardef@e88c77org.highwire.dtl.DTLVardef@15de567_HPS_FORMAT_FIGEXP M_FIG C_FIG

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.