Heme ameliorates dextran sodium sulfate-induced colitis through providing intestinal macrophages with noninflammatory profiles.

The local environment is crucial for shaping the identities of tissue-resident macrophages (Mϕs). When hemorrhage occurs in damaged tissues, hemoglobin induces differentiation of anti-inflammatory Mϕs with reparative function. Mucosal bleeding is one of the pathological features of inflammatory bowel diseases. However, the heme-mediated mechanism modulating activation of intestinal innate immune cells remains poorly understood. Here, we show that heme regulates gut homeostasis through induction of Spi-C in intestinal CX3CR1high Mϕs. Intestinal CX3CR1high Mϕs highly expressed Spi-C in a heme-dependent manner, and myeloid lineage-specific Spic-deficient (Lyz2-cre; Spicflox/flox ) mice showed severe intestinal inflammation with an increased number of Th17 cells during dextran sodium sulfate-induced colitis. Spi-C down-regulated the expression of a subset of Toll-like receptor (TLR)-inducible genes in intestinal CX3CR1high Mϕs to prevent colitis. LPS-induced production of IL-6 and IL-1α, but not IL-10 and TNF-α, by large intestinal Mϕs from Lyz2-cre; Spicflox/flox mice was markedly enhanced. The interaction of Spi-C with IRF5 was linked to disruption of the IRF5-NF-κB p65 complex formation, thereby abrogating recruitment of IRF5 and NF-κB p65 to the Il6 and Il1a promoters. Collectively, these results demonstrate that heme-mediated Spi-C is a key molecule for the noninflammatory signature of intestinal Mϕs by suppressing the induction of a subset of TLR-inducible genes through binding to IRF5.

Safety and protective efficacy of Salmonella Pullorum spiC and rfaH deletion rough mutant as a live attenuated DIVA vaccine candidate.

Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease (PD), which is an acute systemic disease, in chickens, and leads to serious economic losses in many developing countries because of its high morbidity and mortality rate in young chicks. The live-attenuated vaccine is considered to be an effective measure to control the Salmonella infection. In addition, the DIVA (differentiation of infected and vaccinated animals) feature without the interference of serological monitoring of Salmonella infection is an important consideration in the development of the Salmonella vaccine. In this study, we evaluated the immunogenicity and protective efficacy of a S. Pullorum rough mutant S06004ΔspiCΔrfaH as a live attenuated DIVA vaccine candidate in chickens. The S06004ΔspiCΔrfaH exhibited a significant rough lipopolysaccharides (LPS) phenotype which was agglutinated with the acriflavine, not with the O9 mono antibody. Compared to the wild-type, 50% lethal dose (LD50) of the rough mutant increased 100-fold confirmed its attenuation. The mutant strain also showed a decreased bacterial colonization in the spleen and liver. The immunization with the mutant strain had no effect on the body weight and no tissue lesions were observed in the liver and spleen. The high level of the S. Pullorum-specific IgG titers in the serum indicated that significant humoral immune responses were induced in the immunization group. The cellular immune responses were also elicited from the analysis of lymphocyte proliferation and expression of cytokines in the spleen. In addition, the S06004ΔspiCΔrfaH immunized group exhibited a negative response for the serological test, while the wild-type S06004 infection group was strongly positive for the serological test showing a DIVA capability. The survival rates in the vaccinated chickens were 87% after intramuscular challenge with wild-type S. Pullorum, while the survival rates were 20% in the control groups. Overall, these results have demonstrated that the rough mutant S06004ΔspiCΔrfaH strain can be developed as an efficient live attenuated DIVA vaccine candidate to control the systemic S. Pullorum infection without the interference of salmonellosis monitoring program in poultry.

Safety of the Salmonella enterica serotype Dublin strain Sdu189-derived live attenuated vaccine-A pilot study.

Salmonella enterica serovar Dublin (S. Dublin) is an important zoonotic pathogen with high invasiveness. In the prevention and control of the Salmonella epidemic, the live attenuated vaccine plays a very important role. To prevent and control the epidemic of S. Dublin in cattle farms, the development of more effective vaccines is necessary. In this study, we constructed two gene deletion mutants, Sdu189ΔspiC and Sdu189ΔspiCΔaroA, with the parental strain S. Dublin Sdu189. The immunogenicity and protective efficacy were evaluated in the mice model. First, both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for specific pathogen-free (SPF) 6-week-old female BALB/c mice. Second, the specific IgG antibody level and the expression level of cytokine TNF-α, IFN-γ, IL-4, and IL-18 were increased significantly in the vaccinated mice compared to the control group. In addition, the deletion strains were cleared rapidly from organs of immunized mice within 14 d after immunization, while the parental strain could still be detected in the spleen and liver after 21 d of infection. Compared with the parental strain infected group, no obvious lesions were detected in the liver, spleen, and cecum of the deletion strain vaccinated groups of mice. Immunization with Sdu189ΔspiC and Sdu189ΔspiCΔaroA both provided 100% protection against subsequent challenges with the wild-type Sdu189 strain. These results demonstrated that these two deletion strains showed the potential as live attenuated vaccines against S. Dublin infection. The present study established a foundation for screening a suitable live attenuated Salmonella vaccine.

Evaluation of Safety and Protective Efficacy of a waaJ and spiC Double Deletion Korean Epidemic Strain of Salmonella enterica Serovar Gallinarum.

With an aim to develop a highly attenuated and strongly immunogenic distinguishable vaccine candidate, a waaJ (a gene involved in the synthesis of lipopolysaccharide) and spiC (a virulence gene) double deletion Korean epidemic strain of S. enterica ser. Gallinarum (SG005) was constructed. Our results showed that the growth and biochemical characteristics were not altered by this double deletion. The double deletion strain contained dual markers. One was a bacteriological marker (rough phenotype) and the other was a serological marker helping distinguish infected chickens from vaccinated chickens. The double deletion strain showed good genetic stability and reduced resistance to environmental stresses in vitro; furthermore, it was extremely safe and highly avirulent in broilers. Single intramuscular or oral immunization of 7-day-old broilers with the double deletion strain could stimulate the body to produce antibody levels similar to the conventional vaccine strain SG9R. In addition, against a lethal wild-type challenge, it conferred effective protection that was comparable to that seen in the group vaccinated with SG9R. In conclusion, this double deletion strain may be an effective vaccine candidate for controlling S. enterica ser. Gallinarum infection in broilers.

Lineage-instructive functions of the E26-transformation-specific-family transcription factor Spi-C in immune cell development and disease.

Cell fate decisions during hematopoiesis are the consequence of a complex mixture of inputs from cell-intrinsic and cell-extrinsic factors. In rare cases, expression of a single transcription factor, or a few key factors, may be sufficient to dictate lineage differentiation in a precursor cell. The E26-transformation-specific-family transcription factor Spi-C has emerged as an example of a lineage-instructive factor involved in the generation of mature, specialized subsets of both myeloid and lymphoid cells. Spi-C can instruct differentiation of splenic precursors into red pulp macrophages responsible for phagocytosing senescent red blood cells. In the B cell compartment, Spi-C acts as a key regulator of cell fate decisions at the pro-B to pre-B cell stage and for plasma cell differentiation. Spi-C regulates key genes including Nfkb1, Bach2, Syk, and Blnk to regulate cell cycle entry and B cell differentiation. Here, we review the biology of the lineage-instructive transcription factor Spi-C and its contribution to mechanisms of disease in macrophages and B cells. This article is categorized under: Cancer > Molecular and Cellular Physiology Immune System Diseases > Molecular and Cellular Physiology Infectious Diseases > Genetics/Genomics/Epigenetics.

WbaP is required for swarm motility and intramacrophage multiplication of Salmonella Enteritidis spiC mutant by glucose use ability.

Salmonella spp. can survive and replicate in macrophage cells to cause persistent infection, SpiC is a necessary T3SS effector, but its pathogenic mechanism is still not known completely. In our study, Salmonella Enteritidis spiC mutant (SEΔspiC) was found to have stronger swarming motility and intramacrophage hyperproliferation which was closely related to glucose metabolism. SEΔspiC wbaP::Tn5 mutant was screened out by transposon mutagenesis, which had weaker swarming motility and intramacrophage replication ability than SEΔspiC in the presence of glucose. Bioinformatics displayed that undecaprenyl-phosphate galactose phosphotransferase (Wbap), encoded by wbaP gene, was a key enzyme for glucose metabolism and Lipopolysaccharide(LPS) synthesis, which confirmed our outcome that Wbap was involved in intramacrophage replication ability by glucose use in addition to swarming motility based on SEΔspiC. This discovery will further promote the understanding of the interaction between wbaP gene and spiC gene and the intracellular Salmonella replication mechanism.

Prolonged fasting drives a program of metabolic inflammation in human adipose tissue.

OBJECTIVE: The human adaptive fasting response enables survival during periods of caloric deprivation. A crucial component of the fasting response is the shift from glucose metabolism to utilization of lipids, underscoring the importance of adipose tissue as the central lipid-storing organ. The objective of this study was to investigate the response of adipose tissue to a prolonged fast in humans. METHODS: We performed RNA sequencing of subcutaneous adipose tissue samples longitudinally collected during a 10-day, 0-calorie fast in humans. We further investigated observed transcriptional signatures utilizing cultured human monocytes and Thp1 cells. We examined the cellularity of adipose tissue biopsies with transmission electron microscopy and tested for associated changes in relevant inflammatory mediators in the systemic circulation by ELISA assays of longitudinally collected blood samples. RESULTS: Coincident with the expected shift away from glucose utilization and lipid storage, we demonstrated downregulation of pathways related to glycolysis, oxidative phosphorylation, and lipogenesis. The canonical lipolysis pathway was also downregulated, whereas fasting drove alternative lysosomal paths to lipid digestion. Unexpectedly, the dominant induced pathways were associated with immunity and inflammation, although this only became evident at the 10-day time point. Among the most augmented transcripts were those associated with macrophage identity and function, such as members of the erythroblast transformation-specific (ETS) transcription factor family. Key components of the macrophage transcriptional signal in fasting adipose tissue were recapitulated with induced expression of two of the ETS transcription factors via cultured macrophages, SPIC and SPI1. The inflammatory signal was further reflected by an increase in systemic inflammatory mediators. CONCLUSIONS: Collectively, this study demonstrates an unexpected role of metabolic inflammation in the human adaptive fasting response.

Opposing Roles for the Related ETS-Family Transcription Factors Spi-B and Spi-C in Regulating B Cell Differentiation and Function.

Generation of specific antibodies during an immune response to infection or vaccination depends on the ability to rapidly and accurately select clones of antibody-secreting B lymphocytes for expansion. Antigen-specific B cell clones undergo the cell fate decision to differentiate into antibody-secreting plasma cells, memory B cells, or germinal center B cells. The E26-transformation-specific (ETS) transcription factors Spi-B and Spi-C are important regulators of B cell development and function. Spi-B is expressed throughout B cell development and is downregulated upon plasma cell differentiation. Spi-C is highly related to Spi-B and has similar DNA-binding specificity. Heterozygosity for Spic rescues B cell development and B cell proliferation defects observed in Spi-B knockout mice. In this study, we show that heterozygosity for Spic rescued defective IgG1 secondary antibody responses in Spib-/- mice. Plasma cell differentiation was accelerated in Spib-/- B cells. Gene expression, ChIP-seq, and reporter gene analysis showed that Spi-B and Spi-C differentially regulated Bach2, encoding a key regulator of plasma cell and memory B cell differentiation. These results suggest that Spi-B and Spi-C oppose the function of one another to regulate B cell differentiation and function.

Counter Regulation of Spic by NF-κB and STAT Signaling Controls Inflammation and Iron Metabolism in Macrophages.

Activated macrophages must carefully calibrate their inflammatory responses to balance efficient pathogen control with inflammation-mediated tissue damage, but the molecular underpinnings of this "balancing act" remain unclear. Using genetically engineered mouse models and primary macrophage cultures, we show that Toll-like receptor (TLR) signaling induces the expression of the transcription factor Spic selectively in patrolling monocytes and tissue macrophages by a nuclear factor κB (NF-κB)-dependent mechanism. Functionally, Spic downregulates pro-inflammatory cytokines and promotes iron efflux by regulating ferroportin expression in activated macrophages. Notably, interferon-gamma blocks Spic expression in a STAT1-dependent manner. High levels of interferon-gamma are indicative of ongoing infection, and in its absence, activated macrophages appear to engage a "default" Spic-dependent anti-inflammatory pathway. We also provide evidence for the engagement of this pathway in sterile inflammation. Taken together, our findings uncover a pathway wherein counter-regulation of Spic by NF-κB and STATs attune inflammatory responses and iron metabolism in macrophages.

Controversy Surrounding the Function of SpiC Protein in Salmonella: An Overview.

Salmonella is an important pathogenic microorganism that can infect humans and animals and has been studied globally as a model microorganism for its pathogenesis. The SpiC protein of T3SS2 is a significant factor that has been studied for almost 20 years, but to date, the function/effect of SpiC in the pathogenesis of Salmonella has not been completely understood. There is controversy over the functions of SpiC protein in the literature. Thus, an overview of the literature on SpiC protein is provided here which highlights expression features of SpiC protein and its various functions and effect.

Evaluation of the Safety and Protection Efficacy of spiC and nmpC or rfaL Deletion Mutants of Salmonella Enteritidis as Live Vaccine Candidates for Poultry Non-Typhoidal Salmonellosis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a host-ranged pathogen that can infect both animals and humans. Poultry and poultry products are the main carriers of S. Enteritidis, which can be transmitted to humans through the food chain. To eradicate the prevalence of S. Enteritidis in poultry farms, it is necessary to develop novel vaccines against the pathogen. In this study, we constructed two vaccine candidates, CZ14-1∆spiC∆nmpC and CZ14-1∆spiC∆rfaL, and evaluated their protective efficacy. Both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for three-day-old specific-pathogen free (SPF) White Leghorns and Hyline White chickens. Immunization with the mutant candidates induced highly specific humoral immune responses and expression of cytokines IFN-γ, IL-1ß, and IL-6. In addition, the mutant strains were found to be persistent for almost three weeks post-infection. The survival percentages of chickens immunized with CZ14-1∆spiC∆nmpC and CZ14-1∆spiC∆rfaL reached 80% and 75%, respectively, after challenge with the parental strain. Overall, these results demonstrate that the two mutant strains can be developed as live attenuated vaccines.

RAG-Mediated DNA Breaks Attenuate PU.1 Activity in Early B Cells through Activation of a SPIC-BCLAF1 Complex.

Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development.

GSMC: Combining Parallel Gibbs Sampling with Maximal Cliques for Hunting DNA Motif.

Regulatory elements are responsible for regulating gene transcription. Therefore, identification of these elements is a tremendous challenge in the field of gene expression. Transcription factors (TFs) play a key role in gene regulation by binding to target promoter sequences. A set of conserved sequence patterns with a highly similar structure that is bound by a TF is called a motif. Motif discovery has been a difficult problem over the past decades. Meanwhile, it is a foundation stone in meeting this challenge. Recent advances in obtaining genomic sequences and high-throughput gene expression analysis techniques have enabled the rapid development of computational methods for motif discovery. As a result, a large number of motif-finding algorithms aiming at various motif models have sprung up in the past few years. However, most of them are not suitable for analysis of the large data sets generated by next-generation sequencing. To better handle large-scale ChIP-Seq data and achieve better performance in computational time and motif detection accuracy, we propose an excellent motif-finding algorithm known as GSMC (Combining Parallel Gibbs Sampling with Maximal Cliques for hunting DNA Motif). The GSMC algorithm consists of two steps. First, we employ the commonly used Gibbs sampling to generating initial motifs. Second, we utilize maximal cliques to cluster motifs according to Similarity with Position Information Contents (SPIC). Consequently, we raise the detection accuracy in a great degree, in the meantime holding comparative computation efficiency. In addition, we can find much more credible cofactor interacting motifs.

The Heme Connection: Linking Erythrocytes and Macrophage Biology.

Erythroid function and development is intimately linked to macrophages. The primary function of erythrocytes is oxygen delivery, which is mediated by iron-containing hemoglobin. The major source of this iron is a recycling pathway where macrophages scavenge old and damaged erythrocytes to release iron contained within the heme moiety. Macrophages also promote erythropoiesis by providing a supportive niche in the bone marrow as an integral component of "erythorblastic islands." Importantly, inflammation leads to alterations in iron handling by macrophages with significant impact on iron homeostasis and erythropoiesis. The importance of macrophages in erythropoiesis and iron homeostasis is well established and has been extensively reviewed. However, this developmental relationship is not one way, and erythrocytes can also regulate macrophage development and function. Erythrocyte-derived heme can induce the development of iron-recycling macrophages from monocytes, engage pattern recognition receptors to activate macrophages, and act as ligand for specific nuclear receptors to modulate macrophage function. Here, we discuss the role of heme as a signaling molecule impacting macrophage homeostasis. We will review these actions of heme within the framework of our current understanding of the role of micro-environmental factors in macrophage development and function.

Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine.

Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease.