Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

Transcriptome analysis of Kunming mice responses to the bite of Xenopsylla cheopis.

BACKGROUND: Flea bites could trigger a series of complex molecular responses in the host. However, our understanding of the responses at the molecular level is still relatively limited. This study quantifies the changes in gene expression in mice after flea bites by RNA sequencing (RNA-seq) from their spleens, revealing the potential biological effects of host response to flea bites. METHODS: RNA-seq was used for transcriptome analysis to screen for differentially expressed genes (DEGs) between the control mice group and the flea bite mice group. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on DEGs. Protein-protein interaction (PPI) network analysis on DEGs related to immune processes was performed. Finally, we randomly selected several genes from the screened DEGs to validate the results from the transcriptome data by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: A total of 521 DEGs were identified, including 277 upregulated and 244 downregulated. There were 258 GO terms significantly enriched by upregulated DEGs and 419 GO terms significantly enriched by downregulated DEGs. Among the upregulated DEGs, 22 GO terms were associated with immune cells (e.g., B cells and T cells) and immune regulatory processes, while among the downregulated DEGs, 58 GO terms were associated with immune cells and immune regulatory processes. Through PPI analysis, we found that CD40 molecules with significantly downregulated expression levels after flea bites may play an important role in host immune regulation. Through KEGG pathway enrichment analysis, a total of 26 significantly enriched KEGG pathways were identified. The RT-qPCR analysis results indicated that the transcriptome sequencing results were reliable. CONCLUSIONS: Through in-depth analysis of transcriptome changes in mice caused by flea bites, we revealed that flea bites could stimulate a series of biological and immunological responses in mice. These findings not only provided a deeper understanding of the impact of flea bites on the host but also provided a basis for further research on the interaction between ectoparasites and the host. We believe that digging deeper into the significance of these transcriptome changes will help reveal more about the adaptive response of the host to ectoparasites.

Immunological, histological and immunohistochemical alternations induced by zinc oxide nanoparticles and mureer plant in spleen albino rats with the prospective anti-inflammatory action of gallic acid.

The current study was proposed to evaluate the mortal impacts of either alone or mixed treatments of zinc oxide nanoparticles (ZnO NPs) and mureer or Senecio glaucus L. plant (SP) on spleen tissue via immunological and histological studies and to estimate the likely immunomodulatory effect of gallic acid (GA) for 30 days in rats. Rats were classified into eight groups with orally treated: Control, GA (100mg/kg), ZnO NPs (150mg/kg), SP (400mg/kg), GA+ZnO NPs (100,150mg/kg), GA+SP (100,400mg/kg), ZnONPs+SP (150,400mg/kg) and GA+ZnONPs+SP (100,150,400mg/kg). Interleukin-6 (IL-6) level was measured using an enzyme-linked immunoassay (ELISA). Also, the pro-apoptotic protein (caspase-3) expression was estimated using an immunohistochemistry assay. Our data revealed that ZnO NPs and SP triggered a significant increase in the levels of IL-6 and total lipids (TL) and the activity of lactate dehydrogenase (LDH), (p<0.001). Furthermore, they overexpressed caspase-3 and caused lymphoid depletion. They revealed that the immunotoxic outcome of mixed treatment was more than the outcome of the alone treatment. However, GA restored the spleen damage from these adverse results. Finally, this study indicated that ZnO NPs and SP might be immunotoxic and splenotoxic agents; however, GA may be displayed as an anti-inflammatory and splenic-protective agent.

Unswitched memory B cell deficiency in children with sickle cell disease and response to pneumococcal polysaccharide vaccine.

Early mortality in sickle cell disease (SCD) is attributed to increased infections due to loss of splenic function. Marginal zone B cells are important for initial opsonization of pathogens and can be absent in spleen histopathology in SCD. The frequency of unswitched memory B cells (UMBC), the circulating correlate of marginal zone B cells, reflects the immunologic function of the spleen. We hypothesized that asplenia in SCD is associated with alterations in the peripheral blood lymphocyte population and explored whether UMBC deficiency was associated with a clinical phenotype. We analyzed B cell subsets and clinical history for 238 children with SCD and 63 controls. The median proportion of UMBCs was lower in children with SCD compared with controls (4.7% vs. 6.6%, p < .001). Naïve B cells were higher in SCD compared with controls (80.6 vs. 76.3%, respectively, p = .02). UMBC frequency declined by 3.4% per year increase in age in SCD (95% CI: 2%, 4.7%, p < .001), but not in controls. A majority of children in all cohorts had an IgM concentration in the normal range for age and there were no differences between groups (p = .13). Subjects developed titers adequate for long-term protection to fewer serotypes in the polysaccharide vaccine than controls (14.7 vs. 19.4, p < .001). In this cohort, bacteremia was rare and specific clinical complications were not associated with UMBC proportion. In summary, UMBC deficiency occurs in SCD and is associated with age. Future studies should investigate B cell subsets prospectively and identify the mechanism of B cell loss in the spleen.

Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

The compensatory role of T cells from lymph nodes in mice with splenectomy.

The spleen is a vital organ for the immune system, while splenectomy may be necessary for various reasons. However, there is limited research on the impact of splenectomy on T cell function in peripheral lymph nodes as a compensatory mechanism in preventing infections. This study aimed to investigate the characteristics and function of CD8+ and CD4+ T cells in different peripheral lymph nodes during viral infection using a well-established splenectomy model. The results revealed that splenectomy caused an increase in CD8+GP33+ T cells in the mesenteric lymph nodes (MLN). Moreover, we demonstrated that splenectomy resulted in an increase of effector KLRG1+ T cells in the MLN. Additionally, the number of CD4+ cytotoxic T cells (CD4 CTLs) was also elevated in the peripheral lymph nodes of mice with splenectomy. Surprisingly, aged mice exhibited a stronger compensatory ability than adult mice, as evidenced by an increase in effector CD8+ T cells in all peripheral lymph nodes. These findings provide compelling evidence that T cells in MLN play a crucial role in protecting individuals with splenectomy against viral infections. The study offers new insights into understanding the changes in the immune system of individuals with splenectomy and highlights the potential compensatory mechanisms involved by T cells in peripheral lymph nodes.

Yogurt Alleviates Cyclophosphamide-Induced Immunosuppression in Mice through D-Lactate.

Numerous studies have investigated the immunomodulatory effects of yogurt, but the underlying mechanism remained elusive. This study aimed to elucidate the alleviating properties of yogurt on immunosuppression and proposed the underlying mechanism was related to the metabolite D-lactate. In the healthy mice, we validated the safety of daily yogurt consumption (600 µL) or D-lactate (300 mg/kg). In immunosuppressed mice induced by cyclophosphamide (CTX), we evaluated the immune regulation of yogurt and D-lactate. The result showed that yogurt restored body weight, boosted immune organ index, repaired splenic tissue, recovered the severity of delayed-type hypersensitivity reactions and increased serum cytokines (IgA, IgG, IL-6, IFN-γ). Additionally, yogurt enhanced intestinal immune function by restoring the intestinal barrier and upregulating the abundance of Bifidobacterium and Lactobacillus. Further studies showed that D-lactate alleviated immunosuppression in mice mainly by promoting cellular immunity. D-lactate recovered body weight and organ development, elevated serum cytokines (IgA, IgG, IL-6, IFN-γ), enhanced splenic lymphocyte proliferation and increased the mRNA level of T-bet in splenic lymphocyte to bolster Th1 differentiation. Finally, CTX is a chemotherapeutic drug, thus, the application of yogurt and D-lactate in the tumor-bearing mouse model was initially explored. The results showed that both yogurt (600 µL) and D-lactate (300 mg/kg) reduced cyclophosphamide-induced immunosuppression without promoting tumor growth. Overall, this study evaluated the safety, immune efficacy and applicability of yogurt and D-lactate in regulating immunosuppression. It emphasized the potential of yogurt as a functional food for immune regulation, with D-lactate playing a crucial role in its immunomodulatory effects.

Anti-inflammatory role of APRIL by modulating regulatory B cells in antigen-induced arthritis.

APRIL (A Proliferation-Inducing Ligand), a member of the TNF superfamily, was initially described for its ability to promote proliferation of tumor cells in vitro. Moreover, this cytokine has been related to the pathogenesis of different chronic inflammatory diseases, such as rheumatoid arthritis. This study aimed to evaluate the ability of APRIL in regulating B cell-mediated immune response in the antigen-induced arthritis (AIA) model in mice. AIA was induced in previously immunized APRIL-transgenic (Tg) mice and their littermates by administration of antigen (mBSA) into the knee joints. Different inflammatory cell populations in spleen and draining lymph nodes were analyzed using flow cytometry and the assay was performed in the acute and chronic phases of the disease, while cytokine levels were assessed by ELISA. In the acute AIA, APRIL-Tg mice developed a less severe condition and a smaller inflammatory infiltrate in articular tissues when compared with their littermates. We also observed that the total cellularity of draining lymph nodes was decreased in APRIL-Tg mice. Flow cytometry analysis revealed an increase of CD19+IgM+CD5+ cell population in draining lymph nodes and an increase of CD19+CD21hiCD23hi (B regulatory) cells in APRIL-Tg mice with arthritis as well as an increase of IL-10 and CXCL13 production in vitro.

Inhibition of caspase pathways limits CD4+ T cell loss and restores host anti-retroviral function in HIV-1 infected humanized mice with augmented lymphoid tissue.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3 L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3 L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.

Dynamic changes in B cell subpopulations in response to triple-negative breast cancer development.

Despite presenting a worse prognosis and being associated with highly aggressive tumors, triple-negative breast cancer (TNBC) is characterized by the higher frequency of tumor-infiltrating lymphocytes, which have been implicated in better overall survival and response to therapy. Though recent studies have reported the capacity of B lymphocytes to recognize overly-expressed normal proteins, and tumor-associated antigens, how tumor development potentially modifies B cell response is yet to be elucidated. Our findings reveal distinct effects of 4T1 and E0771 murine tumor development on B cells in secondary lymphoid organs. Notably, we observe a significant expansion of total B cells and plasma cells in the tumor-draining lymph nodes (tDLNs) as early as 7 days after tumor challenge in both murine models, whereas changes in the spleen are less pronounced. Surprisingly, within the tumor microenvironment (TME) of both models, we detect distinct B cell subpopulations, but tumor development does not appear to cause major alterations in their frequency over time. Furthermore, our investigation into B cell regulatory phenotypes highlights that the B10 Breg phenotype remains unaffected in the evaluated tissues. Most importantly, we identified an increase in CD19 + LAG-3 + cells in tDLNs of both murine models. Interestingly, although CD19 + LAG-3 + cells represent a minor subset of total B cells (< 3%) in all evaluated tissues, most of these cells exhibit elevated expression of IgD, suggesting that LAG-3 may serve as an activation marker for B cells. Corroborating with these findings, we detected distinct cell cycle and proliferation genes alongside LAG-3 analyzing scRNA-Seq data from a cohort of TNBC patients. More importantly, our study suggests that the presence of LAG-3 B cells in breast tumors could be associated with a good prognosis, as patients with higher levels of LAG-3 B cell transcripts had a longer progression-free interval (PFI). This novel insight could pave the way for targeted therapies that harness the unique properties of LAG-3 + B cells, potentially offering new avenues for improving patient outcomes in TNBC. Further research is warranted to unravel the mechanistic pathways of these cells and to validate their prognostic value in larger, diverse patient cohorts.

Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses.

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6 mg/kg VB6), VB6-deprived (0.5 mg/kg VB6) or VB6-enhanced (12 mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5 mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5 mg/kg VB6 groups, and in the 12.5 mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

Ficus hirta Vahl. alleviate LPS induced apoptosis via down-regulating of miR-411 in orange-spotted grouper (Epinephelus coioides) spleen cell.

Ficus hirta Vahl. (FhV) has been shown to have antimicrobial and antiviral efficacy. To further ascertain the pharmacological properties of FhV., and to search for alternatives to antibiotics. An in vitro experiment was carried out to evaluate what influence FhV. would have on LPS-induced apoptosis. In this study, Fas, an apoptosis receptor, was cloned, which included a 5'-UTR of 39 bp, an ORF of 951 bp, a protein of 316 amino acids, and a 3'-UTR of 845 bp. EcFas was most strongly expressed in the spleen tissue of orange-spotted groupers. In addition, the apoptosis of fish spleen cells induced by LPS was concentration-dependent. Interestingly, appropriate concentrations of FhV. alleviated LPS-induced apoptosis. Inhibition of miR-411 further decreased the inhibitory effect of Fas on apoptosis, which reduced Bcl-2 expression and mitochondrial membrane potential, enhanced the protein expression of Bax and Fas. More importantly, the FhV. could activate miR-411 to improve this effect. In addition, luciferase reporter assays showed that miR-411 binds to Fas 3'-UTR to inhibit Fas expression. These findings provide evidence that FhV. alleviates LPS-induced apoptosis by activating miR-411 to inhibit Fas expression and, therefore, provided possible strategies for bacterial infections in fish.

Sex- and species-specific contribution of CD99 to T cell costimulation during multiple sclerosis.

BACKGROUND: Differences in immune responses between women and men are leading to a strong sex bias in the incidence of autoimmune diseases that predominantly affect women, such as multiple sclerosis (MS). MS manifests in more than twice as many women, making sex one of the most important risk factor. However, it is incompletely understood which genes contribute to sex differences in autoimmune incidence. To address that, we conducted a gene expression analysis in female and male human spleen and identified the transmembrane protein CD99 as one of the most significantly differentially expressed genes with marked increase in men. CD99 has been reported to participate in immune cell transmigration and T cell regulation, but sex-specific implications have not been comprehensively investigated. METHODS: In this study, we conducted a gene expression analysis in female and male human spleen using the Genotype-Tissue Expression (GTEx) project dataset to identify differentially expressed genes between women and men. After successful validation on protein level of human immune cell subsets, we assessed hormonal regulation of CD99 as well as its implication on T cell regulation in primary human T cells and Jurkat T cells. In addition, we performed in vivo assays in wildtype mice and in Cd99-deficient mice to further analyze functional consequences of differential CD99 expression. RESULTS: Here, we found higher CD99 gene expression in male human spleens compared to females and confirmed this expression difference on protein level on the surface of T cells and pDCs. Androgens are likely dispensable as the cause shown by in vitro assays and ex vivo analysis of trans men samples. In cerebrospinal fluid, CD99 was higher on T cells compared to blood. Of note, male MS patients had lower CD99 levels on CD4+ T cells in the CSF, unlike controls. By contrast, both sexes had similar CD99 expression in mice and Cd99-deficient mice showed equal susceptibility to experimental autoimmune encephalomyelitis compared to wildtypes. Functionally, CD99 increased upon human T cell activation and inhibited T cell proliferation after blockade. Accordingly, CD99-deficient Jurkat T cells showed decreased cell proliferation and cluster formation, rescued by CD99 reintroduction. CONCLUSIONS: Our results demonstrate that CD99 is sex-specifically regulated in healthy individuals and MS patients and that it is involved in T cell costimulation in humans but not in mice. CD99 could potentially contribute to MS incidence and susceptibility in a sex-specific manner.

The immune system protects us from bacterial and viral infections and impacts the outcome of many diseases. Thus, understanding immunological processes is crucial to unravel pathogenic mechanisms and to develop new therapeutic treatment options. Sex is a biological variable affecting immunity and it is known that females and males differ in their immunological responses. Women mount stronger immune responses leading to more rapid control of infections and greater vaccine efficacy compared to men. However, this enhanced immune responsiveness is accompanied by female preponderance and susceptibility to autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis (MS). MS sex ratio varies around 2:1 to 3:1 with a steadily increasing incidence in female MS patients making sex one of the top risk factors for developing MS. However, the underlying biological mechanisms including sex hormones as well as genetic and epigenetic factors and their complex interplay remain largely unknown. Here, we discovered the gene and its encoded protein CD99 to be differentially expressed between women and men with men showing increased expression on many immune cell subsets including T cells. Since T cells are key contributors to MS pathogenesis, we examined the role of CD99 on T cells of healthy individuals and MS patients. We were able to identify CD99-mediated T cell regulation, which might contribute to sex differences in MS susceptibility and incidence indicating the importance to include sex as a biological variable. Of note, these differences were not reproduced in mice showing the necessity of functional research in humans.

Innervation of nociceptor neurons in the spleen promotes germinal center responses and humoral immunity.

Peripheral sensory neurons widely innervate various tissues to continuously monitor and respond to environmental stimuli. Whether peripheral sensory neurons innervate the spleen and modulate splenic immune response remains poorly defined. Here, we demonstrate that nociceptive sensory nerve fibers extensively innervate the spleen along blood vessels and reach B cell zones. The spleen-innervating nociceptors predominantly originate from left T8-T13 dorsal root ganglia (DRGs), promoting the splenic germinal center (GC) response and humoral immunity. Nociceptors can be activated by antigen-induced accumulation of splenic prostaglandin E2 (PGE2) and then release calcitonin gene-related peptide (CGRP), which further promotes the splenic GC response at the early stage. Mechanistically, CGRP directly acts on B cells through its receptor CALCRL-RAMP1 via the cyclic AMP (cAMP) signaling pathway. Activating nociceptors by ingesting capsaicin enhances the splenic GC response and anti-influenza immunity. Collectively, our study establishes a specific DRG-spleen sensory neural connection that promotes humoral immunity, suggesting a promising approach for improving host defense by targeting the nociceptive nervous system.

NLRP6 negatively regulates host defense against polymicrobial sepsis.

Introduction: Sepsis remains a major cause of death in Intensive Care Units. Sepsis is a life-threatening multi-organ dysfunction caused by a dysregulated systemic inflammatory response. Pattern recognition receptors, such as TLRs and NLRs contribute to innate immune responses. Upon activation, some NLRs form multimeric protein complexes in the cytoplasm termed "inflammasomes" which induce gasdermin d-mediated pyroptotic cell death and the release of mature forms of IL-1ß and IL-18. The NLRP6 inflammasome is documented to be both a positive and a negative regulator of host defense in distinct infectious diseases. However, the role of NLRP6 in polymicrobial sepsis remains elusive. Methods: We have used NLRP6 KO mice and human septic spleen samples to examine the role of NLRP6 in host defense in sepsis. Results: NLRP6 KO mice display enhanced survival, reduced bacterial burden in the organs, and reduced cytokine/chemokine production. Co-housed WT and KO mice following sepsis show decreased bacterial burden in the KO mice as observed in singly housed groups. NLRP6 is upregulated in CD3, CD4, and CD8 cells of septic patients and septic mice. The KO mice showed a higher number of CD3, CD4, and CD8 positive T cell subsets and reduced T cell death in the spleen following sepsis. Furthermore, administration of recombinant IL-18, but not IL-1ß, elicited excessive inflammation and reversed the survival advantages observed in NLRP6 KO mice. Conclusion: These results unveil NLRP6 as a negative regulator of host defense during sepsis and offer novel insights for the development of new treatment strategies for sepsis.

Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis.

ORMDL3 is a prominent member of a family of highly conserved endoplasmic reticulum resident proteins, ORMs (ORM1 and ORM2) in yeast, dORMDL in Drosophila and ORMDLs (ORMDL1, ORMDL2, and ORMDL3) in mammals. ORMDL3 mediates feedback inhibition of de novo sphingolipid synthesis. Expression levels of ORMDL3 are associated with the development of inflammatory and autoimmune diseases including asthma, systemic lupus erythematosus, type 1 diabetes mellitus and others. It has been shown that simultaneous deletions of other ORMDL family members could potentiate ORMDL3-induced phenotypes. To understand the complex function of ORMDL proteins in immunity in vivo, we analyzed mice with single or double deletions of Ormdl genes. In contrast to other single and double knockouts, simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupted blood homeostasis and reduced immune cell content in peripheral blood and spleens of mice. The reduced number of splenocytes was not caused by aberrant immune cell homing. A competitive bone marrow transplantation assay showed that the development of Ormdl1-/-/Ormdl3-/- B cells was dependent on lymphocyte intrinsic factors. Highly increased sphingolipid production was observed in the spleens and bone marrow of Ormdl1-/-/Ormdl3-/- mice. Slight, yet significant, increase in some sphingolipid species was also observed in the spleens of Ormdl3-/- mice and in the bone marrow of both, Ormdl1-/- and Ormdl3-/- single knockout mice. Taken together, our results demonstrate that the physiological expression of ORMDL proteins is critical for the proper development and circulation of lymphocytes. We also show cell-type specific roles of individual ORMDL family members in the production of different sphingolipid species.

A novel vaccine strategy against Brucellosis using Brucella abortus multi-epitope OMPs vaccine based on Lactococcus lactis live bacterial vectors.

Brucella infections typically occur in mucosal membranes, emphasizing the need for mucosal vaccinations. This study evaluated the effectiveness of orally administering Lactococcus lactis (L. lactis) for producing the Brucella abortus multi-epitope OMPs peptide. A multi-epitope plasmid was generated through a reverse vaccinology method, and mice were administered the genetically modified L. lactis orally as a vaccine. The plasmid underwent digestion, synthesizing a 39 kDa-sized protein known as OMPs by the target group. The sera of mice that were administered the pNZ8124-OMPs-L. lactis vaccine exhibited a notable presence of IgG1 antibodies specific to outer membrane proteins (OMPs), heightened levels of interferon (IFN-λ) and tumor necrosis factor alpha (TNF-α), and enhanced transcription rates of interleukin 4 (IL-4) and interleukin 10 (IL-10). The spleen sections from the pNZ8124-OMPs-L. lactis and IRIBA group had less morphological damage associated with inflammation, infiltration of lymphocytes, and lesions to the spleen. The findings present a novel approach to utilizing the food-grade, non-pathogenic L. lactis as a protein cell factory to synthesize innovative immunological candidate OMPs. This approach offers a distinctive way to evaluate experimental medicinal items' practicality, safety, affordability, and long-term sustainability.

ß-Glucan Subverts the Function of Myeloid Cells in Neonates.

ß-Glucan is the main component of the cell wall of pathogen-associated molecular patterns (PAMPs) including various yeast, fungi, or certain bacteria. Previous reports demonstrated that ß-glucan was widely investigated as a potent immunomodulators to stimulate innate and adaptive immune responses, which indicated that it could be recommended as an effective adjuvant in immunotherapy. However, the detailed effects of ß-glucan on neonatal immunity are still largely unknown. Here, we found that ß-glucan did not affect the frequencies and numbers of myeloid cells in the spleen and bone marrow from neonates. Functional assay revealed that ß-glucan from neonates compromised the immunosuppressive function of immature myeloid cells, which were myeloid-derived suppressor cells (MDSCs). Flow cytometry or gene expression analysis revealed that ß-glucan-derived polymorphonuclear (PMN)-MDSCs produced lower level of reactive oxygen species (ROS) and arginase-1 (Arg1) in neonatal mice. Furthermore, ß-glucan administration significantly decreased the frequency and ROS level of PMN-MDSCs in vitro. These observations suggest that ß-glucan facilitates the maturation of myeloid cells in early life, which may contribute to its beneficial effects against immune disorders later in life.

The effect of PDK1 in maintaining immune cell development and function.

3-phosphoinositide-dependent protein kinase 1 (PDK1) exhibits a substantial influence on immune cell development by establishing a vital connection between PI3K and downstream mTOR signaling cascades. However, it remains unclear whether PDK1 signaling affects the homeostasis and functionality of immune cells. To explore the impact of PDK1 on different immune cells within immune organs, transgenic mouse strains with lymphocyte-specific PDK1 knockout (PDK1fl/fl CD2-Cre) were generated. Unlike wild-type (WT) mice, lymphocyte-specific PDK1 knockout (KO) mice exhibited thymic atrophy, elevated percentages of CD8+ T cells and neutrophils, and reduced proportions of γδ T cells, B cells, and NK cells in the spleen. Functional analysis revealed elevated release of IFN-γ and IL-17A by T cells in PDK1 KO mice, contrasting with diminished levels observed in γδ T cells and Treg cells. Furthermore, the activation, cytotoxicity, and migratory potential of γδ T cells in PDK1 KO mice are heightened, indicating a potential association with the regulation of the mTOR signaling pathway. To conclude, the findings of this research demonstrated that specific knockout of PDK1 in lymphocytes hindered T cell development in the thymus and exhibited a substantial influence on immune cell homeostasis in the spleen and lymph nodes.

Therapeutic role of interferon-γ in experimental autoimmune encephalomyelitis is mediated through a tolerogenic subset of splenic CD11b+ myeloid cells.

Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-ß or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-ß and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-ß-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-ß. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.