Antibacterial Immune Response Research Articles

Treatment of Bacterial Infections with β-Lactams: Cooperation with Innate Immunity.

β-Lactams are the most widely prescribed antibiotics used for the control and treatment of bacterial infections. The direct effect of β-lactams on bacteria is well studied worldwide. In the context of infections and as a consequence of their direct activity against the pathogen, β-lactams also regulate antibacterial immune responses. This knowledge has led to the theorem that the effectiveness of β-lactam treatment results from the synergy between the drug and the immune response. Key players in this immune response, with an essential role in the clearance of live and dead bacteria, are the myeloid cells. In this review, we summarize the data that shed light on how β-lactams interact with myeloid cells during bacterial infection treatment.

Drosophila melanogaster larvae are tolerant to oral infection with the bacterial pathogen Photorhabdus luminescens.

The fruit fly Drosophila melanogaster is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The Gram-negative bacterium Photorhabdus luminescens is an insect-specific pathogen that forms a mutualistic relationship with the entomopathogenic nematode Heterorhabditis bacteriophora . Here we find that oral infection of D. melanogaster larvae with P. luminescens moderately reduces their survival ability while the bacteria replicate efficiently in the infected insects. This information will contribute towards understanding host gut immunity against potent bacterial pathogens.

Fabrication of bio-engineered chitosan nanoformulations to inhibition of bacterial infection and to improve therapeutic potential of intestinal microflora, intestinal morphology, and immune response in infection induced rat model

Overdosage of antibiotics used to prevent bacterial infections in the human and animal gastrointestinal tract would result in disturbing of intestinal barrier, significant misbalancing effects of intestinal microflora and persuading bacterial resistance. The main objective of the present investigation is to design and develop novel combinations of organic curcumin (Cur) and antimicrobial peptide (Amp) loaded chitosan nanoformulations (Cur/Amp@CS NPs) to improve significant effects on antibacterial action, immune response, intestine morphology, and intentional microflora. The antibacterial efficiency of the prepared nanoformulations was evaluated using Escherichia coli (E. coli) induced bacterial infections in GUT of Rat models. Further, we studied the cytocompatibility, inflammatory responses, α-diversity, intestinal morphology, and immune responses of treated nanoformulations in rat GUT models. The results indicated that Cur/Amp@CS NPs are greatly beneficial for intestinal microflora and could be a prodigious alternative of antibiotics.

Negative regulation of interferon regulatory factor 6 (IRF6) in interferon and NF-κB signalling pathways of common carp (Cyprinus carpio L.)

BackgroundInterferon (IFN) regulatory factors (IRFs) is a kind of transcription factors, which play an important role in regulating the expression of type I IFN and related genes. In mammals, IRF6 is not relevant with IFN expression, while zebrafish IRF6 was reported to be a positive regulator of IFN expression and could be phosphorylated by both MyD88 and TBK1. However, the role of IRF6 in the immune response and IFN transcription of common carp is unknown.ResultsIn the present study, the cDNA of IRF6 gene (CcIRF6) was cloned from common carp using RACE technique, with a total length of 1905 bp, encoding 471 amino acid residues, which possesses two functional domains of DBD and IAD. Similarity analysis showed that CcIRF6 had more than 50% similarity with IRFs of other vertebrates, and had the highest similarity with grass carp and zebrafish, among which the DBD domain was much more conserved. The phylogenetic analysis showed that CcIRF6 is in the branch of Osteichthyes and has the closest relationship with grass carp. In healthy common carp, the CcIRF6 was expressed in all the examined tissues, with the highest level in the oral epithelium, and the lowest level in the head kidney. After intraperitoneal injection of poly(I:C) or Aeromonas hydrophila, the expression of CcIRF6 increased in spleen, head kidney, foregut and hindgut of common carp. Moreover, poly(I:C), LPS, PGN and flagellin induced the expression of CcIRF6 in peripheral leukocytes and head kidney leukocytes of common carp in vitro. In EPC cells, CcIRF6 inhibited the expression of some IFN-related genes and pro-inflammatory cytokines, and dual luciferase reporter assay showed that CcIRF6 reduced the activity of IFN and NF-κB reporter genes.ConclusionsThe present study suggests that CcIRF6 is involved in the antiviral and antibacterial immune response of common carp, and negatively regulate the expression of IFN and NF-κB signalling pathways, which provides a theoretical basis for the study and prevention of fish disease pathogenesis.

TroTNFSF6, a tumor necrosis factor ligand superfamily member, promotes antibacterial immune response of golden pompano, Trachinotus ovatus

Tumor necrosis factor ligand superfamily member 6 (TNFSF6), also known as FasL/CD95L, is essential for maintaining the body's immune homeostasis. However, the current reports on TNFSF6 in fish are relatively scarce. In the present study, we conducted functional analyses of a TNFSF6 (TroTNFSF6) from the teleost fish golden pompano (Trachinotus ovatus). TroTNFSF6 is composed of 228 amino acids and has a low similarity with other species (9.65%–58.79%). TroTNFSF6 was expressed in the 11 tissues tested and was significantly up-regulated after Edwardsiella tarda infection. In vivo, overexpression of TroTNFSF6 effectively stimulated the AKP and ACP activities, and reduced bacterial infection in fish tissues. Correspondingly, knockdown of TroTNFSF6 expression resulted in increasing bacterial dissemination and colonization in fish tissues. In vitro, recombinant TroTNFSF6 protein promoted the proliferation of T. ovatus head kidney lymphocytes (HKLs), and promoted the apoptosis of murine liver cancer cells (Hepa1-6). The results indicated that TroTNFSF6 plays an important role in the T. ovatus antibacterial immunity. These observations will facilitate the future in-depth study of teleost TNFSF6.

Rosmarinic acid bolsters antibacterial immunity activity of macrophages by up-regulating PINK1/Parkin-mediated mitophagy

This study aims to explore the molecular mechanism through which rosmarinic acid up-regulates mitophagy and enhances antibacterial immunity activity of macrophages. To be specific, RAW264.7 macrophages were treated with rosmarinic acid and then infected with Staphylococcus aureus. The total mRNA and proteins of the cells were then extracted. The mRNA and protein levels of phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1) were detected by q-PCR and Western blot, respectively. Cell mitochondria isolation kit was employed to isolate mitochondria in macrophages. Recruitment of E3 ubiquitin ligase Parkin to mitochondria and the phosphorylation of Parkin were detected by Western blot. Co-immunoprecipitation and laser confocal microscopy were employed to observe the co-localization of PINK1 and Parkin. Mitochondrial division inhibitor 1(Mdivi-1), small interfering RNA(siRNA)-directed gene knockdown, and plate-colony counting were used to detect the levels of inflammatory cytokines and the intracellular antibacterial ability, in an attempt to confirm that rosmarinic acid promotes antibacterial immunity activity of macrophages through strengthening PINK1/Parkin-mediated mitophagy. The results showed that rosmarinic acid up-regulated the mRNA and protein expression of PINK1, promoted the recruitment of Parkin from cytoplasm to mitochondria and the phosphorylation, and enhanced the interaction between PINK1 and Parkin and their co-localization in macrophages. Blocking mitophagy or knocking PINK1 significantly abrogated the promotion of macrophage antibacterial immune response by rosmarinic acid. In summary, rosmarinic acid enhances antibacterial immunity activity of macrophages through up-regulating PINK1/Parkin-mediated mitophagy.

Neutrophilic Pleuritis Is a Severe Complication of Klebsiella pneumoniae Pneumonia in Old Mice.

The pathomechanisms underlying the frequently observed fatal outcome of Klebsiella pneumoniae pneumonia in elderly patients are understudied. In this study, we examined the early antibacterial immune response in young mice (age 2-3 mo) as compared with old mice (age 18-19 mo) postinfection with K. pneumoniae. Old mice exhibited significantly higher bacterial loads in lungs and bacteremia as early as 24 h postinfection compared with young mice, with neutrophilic pleuritis nearly exclusively developing in old but not young mice. Moreover, we observed heavily increased cytokine responses in lungs and pleural spaces along with increased mortality in old mice. Mechanistically, Nlrp3 inflammasome activation and caspase-1-dependent IL-1β secretion contributed to the observed hyperinflammation, which decreased upon caspase-1 inhibitor treatment of K. pneumoniae-infected old mice. Irradiated old mice transplanted with the bone marrow of young mice did not show hyperinflammation or early bacteremia in response to K. pneumoniae. Collectively, the accentuated lung pathology observed in K. pneumoniae-infected old mice appears to be due to regulatory defects of the bone marrow but not the lung, while involving dysregulated activation of the Nlrp3/caspase-1/IL-1β axis.

Molecular Characterization of a B Cell Adaptor for Phosphoinositide 3-Kinase Homolog in Lamprey (Lampetra japonica) and Its Function in the Immune Response.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.

Senescence of the immune defences and reproductive trade-offs in females of the mealworm beetle, Tenebrio molitor

In the theory of ageing, it has been assumed that ageing is associated with a decline in somatic defences, including the immune system, as a consequence of a trade-off with reproduction. While overall immunity suffers from age-related deterioration (immune senescence), the different components of the immune response appear to age differently. It is also likely that investment among the many arms of the immune system and reproduction with age is finely adjusted to the organisms' reproductive strategy. We investigated this possibility in females of Tenebrio molitor, a species of long-lived insect with reproductive strategies similar to those of long-lived mammals. We specifically tested the effects of immunological challenges imposed early or late in adult life on immune pathway activation as well as fertility early and late in life. We found complex patterns of changes in immune defences with age and age-specific immune challenges with contrasted relationships with female reproduction. While cellular and enzymatic defences showed signs of ageing, they did not trade-off with reproduction. By contrast, the induced antibacterial immune response was found to be unaffected by age and to be highly connected to female fecundity. These findings suggest that these immunological pathways have different functions with regard to female ageing in this insect species.

Bacterial induced miR-144-5p modulates intestinal inflammatory response of Japanese flounder by targeting Hsp90α-dependent NLR signaling pathway

Pathogens infection could induce intestinal inflammatory processes, appropriate intestinal inflammation response had beneficial effects in fish, while excessive inflammation alters enteroendocrine signaling, leading to deregulated immune response. Recently, many researches revealed that microRNAs in teleost serve as vital regulators in innate immunity responses against pathogen infection. However, there are few studies on miRNAs mediated regulatory networks of intestinal inflammatory response in teleost. In our study, miR-144-5p was identified as negative regulators in teleost against bacterial infection. First, we found that the expression of miR-144-5p in flounder both in vivo and in vitro was significantly up-regulated, while the expression of Hsp90α, NLRP3, Caspase1 and IL-1β were significantly down-regulated upon the stimulation of Edwardsiella tarda and lipopolysaccharide (LPS). Furthermore, we confirmed that miR-144-5p modulates intestinal inflammatory responses of flounder by targeting Hsp90α-dependent NLR signaling pathway, in which the miR-144-5p-Hsp90α-NLRP3 signaling pathway was constructed. Besides, the miR-144-5p-Hsp90α-NLRP3 signaling pathway could inhibit antibacterial immune response of flounder and promote the survival of E. tarda. Moreover, the study demonstrated that the miR-144-5p-Hsp90α regulatory network was widely existed in other teleost and human. In conclusion, the expression of miR-144-5p was significantly up-regulated after challenged by E. tarda and LPS, which then down-regulated the expression of inflammation-related cytokines of NLRP3, Caspase1 and IL-1β by targeting Hsp90α, and ultimately promoted bacterial infection. Our findings constructed a novel miR-144-5p-Hsp90α-NLRP3 signaling pathway, which enriched the regulatory network of miRNA on fish intestinal mucosal immunity and would benefit the development of fish disease therapeutic strategies to prevent potentially lethal bacterial infection.

Alterations of the Mucosal Immune Response and Microbial Community of the Skin upon Viral Infection in Rainbow Trout (Oncorhynchus mykiss).

The skin is the largest organ on the surface of vertebrates, which not only acts as the first line of defense against pathogens but also harbors diverse symbiotic microorganisms. The complex interaction between skin immunity, pathogens, and commensal bacteria has been extensively studied in mammals. However, little is known regarding the effects of viral infection on the skin immune response and microbial composition in teleost fish. In this study, we exposed rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus (IHNV) by immersion infection. Through pathogen load detection and pathological evaluation, we confirmed that IHNV successfully invaded the rainbow trout, causing severe damage to the epidermis of the skin. qPCR analyses revealed that IHNV invasion significantly upregulated antiviral genes and elicited strong innate immune responses. Transcriptome analyses indicated that IHNV challenge induced strong antiviral responses mediated by pattern recognition receptor (PRR) signaling pathways in the early stage of the infection (4 days post-infection (dpi)), and an extremely strong antibacterial immune response occurred at 14 dpi. Our 16S rRNA sequencing results indicated that the skin microbial community of IHNV-infected fish was significantly richer and more diverse. Particularly, the infected fish exhibited a decrease in Proteobacteria accompanied by an increase in Actinobacteria. Furthermore, IHNV invasion favored the colonization of opportunistic pathogens such as Rhodococcus and Vibrio on the skin, especially in the later stage of infection, leading to dysbiosis. Our findings suggest that IHNV invasion is associated with skin microbiota dysbiosis and could thus lead to secondary bacterial infection.

LncRNA MIR144HG-derived miR-144 suppresses antibacterial signaling and facilitates bacteria escape in fish

Growing number studies show that lncRNA is an important regulatory factor and plays an irreplaceable role in innate immunity. However, most studies on lncRNA have focused on mammals and it is unknown in lower vertebrates. In this study, we identify a new lncRNA, named MIR144HG, which can have a negative role in antibacterial immunity of Miichthys miiuy caused by Vibrio anguillarum and V. harveyi. MyD88, TAK1 and p65 are very important pathway genes in antibacterial innate immune responses, whether in mammals or lower vertebrates. Here, we found that miR-144–3p could target MyD88, TAK1 and p65, inhibit host antibacterial response and promote bacterial escape. Further studies found that miR-144–3p can be derived by MIR144HG, and finally enhance the inhibitory effect of miR-144–3p on host antibacterial immunity. Moreover, V. anguillarum and V. harveyi are the two most susceptible Gram-negative pathogens in aquaculture, and the economic losses caused by these two bacteria are immeasurable every year. Our results not only elucidate the mechanism of the lncRNA-miRNA-mRNA axis in antibacterial immune responses, but also provide new insights for understanding the impact of lncRNA on host immunity and bacteria escape.

IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

Utilization of specialized Th1 cells to resist intracellular pathogenic infection represents an important innovation of adaptive immunity. Although transcriptional evidence indicates the potential presence of Th1-like cells in some fish species, the existence of CD3+CD4+IFN-γ+ T cells, their detailed functions, and the mechanism determining their differentiation in these early vertebrates remain unclear. In the present study, we identified a population of CD3+CD4-1+IFN-γ+ (Th1) cells in Nile tilapia upon T-cell activation in vitro or Edwardsiella piscicida infection in vivo. By depleting CD4-1+ T cells or blocking IFN-γ, Th1 cells and their produced IFN-γ were found to be essential for tilapia to activate macrophages and resist the E. piscicida infection. Mechanistically, activated T cells of tilapia produce IL-2, which enhances the STAT5 and mTORC1 signaling that in turn trigger the STAT1/T-bet axis-controlled IFN-γ transcription and Th1 cell development. Additionally, mTORC1 regulates the differentiation of these cells by promoting the proliferation of CD3+CD4-1+ T cells. Moreover, IFN-γ binds to its receptors IFNγR1 and IFNγR2 and further initiates a STAT1/T-bet axis-mediated positive feedback loop to stabilize the Th1 cell polarization in tilapia. These findings demonstrate that, prior to the emergence of tetrapods, the bony fish Nile tilapia had already evolved Th1 cells to fight intracellular bacterial infection, and support the notion that IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to determine Th1 cell fate, which is an ancient mechanism that has been programmed early during vertebrate evolution. Our study is expected to provide novel perspectives into the evolution of adaptive immunity.

Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1β (T. nigroviridis IL-1β). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1β after activation.

Cloning of Toll-like Receptor 3 Gene from Schizothorax prenanti (SpTLR3), and Expressions of Seven SpTLRs and SpMyD88 after Lipopolysaccharide Induction.

Toll-like receptor 3 (SpTLR3) from Schizothorax prenanti (S. prenanti) was cloned and identified, and the tissue distribution of the SpTLR3 gene was examined in this study. Moreover, the relative mRNA expression levels of myeloid differentiation factor 88 gene (SpMyD88) and seven TLR genes (SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3) from S. prenanti after lipopolysaccharide (LPS) challenge were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR). The full length of SpTLR3 gene is 3097 bp, and complete coding sequence (CDS) is 2715 bp, which encodes 904 amino acids. The SpTLR3 amino acid sequence shared 43.94–100% identity with TLR3 sequences from other vertebrates; SpTLR3 was expressed in all eight tissues examined; and the highest level appeared in the liver, which was significantly higher than in all other tissues (p < 0.05), followed by the levels in the heart and muscles. LPS significantly up-regulated all eight genes in the S. prenanti tissues at 12 or 24 h (p < 0.05). Compared with the PBS control group, no significant transcripts changes were found in SpTLR2 or SpTLR3 at 12 h after LPS induction, but they were significantly up-regulated at 24 h (p < 0.001). The most abundant transcripts were found in the head kidney SpTLR22 genes after 24 h LPS induction, with high to low levels, which were SpTLR22-1 (564-fold), SpTLR22-3 (508-fold) and SpTLR22-2 (351-fold). Among these eight genes, the expression level of SpTLR4 was the least up-regulated. Overall, SpTLR4 in the head kidney was involved in the antibacterial immune response earlier, and the level was increased at 12 h with extreme significance after LPS stimulation (p < 0.001), while the other seven genes were the most significantly up-regulated at 24 h post injection. Taken together, the results suggest that SpMyD88, SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3 participate in an innate immune response stimulated by LPS, and the response intensity of the genes was organ-specific, with differing kinetics. Our findings will contribute to a more complete understanding of the roles of these TLR genes in antibacterial immunity.

Loss of Vhl alters trabecular bone loss during S. aureus osteomyelitis in a cell-specific manner.

Osteomyelitis, or bone infection, is a major complication of accidental trauma or surgical procedures involving the musculoskeletal system. Staphylococcus aureus is the most frequently isolated pathogen in osteomyelitis and triggers significant bone loss. Hypoxia-inducible factor (HIF) signaling has been implicated in antibacterial immune responses as well as bone development and repair. In this study, the impact of bone cell HIF signaling on antibacterial responses and pathologic changes in bone architecture was explored using genetic models with knockout of either Hif1a or a negative regulator of HIF-1α, Vhl. Deletion of Hif1a in osteoblast-lineage cells via Osx-Cre (Hif1aΔOB ) had no impact on bacterial clearance or pathologic changes in bone architecture in a model of post-traumatic osteomyelitis. Knockout of Vhl in osteoblast-lineage cells via Osx-Cre (VhlΔOB ) caused expected increases in trabecular bone volume per total volume (BV/TV) at baseline and, intriguingly, did not exhibit an infection-mediated decline in trabecular BV/TV, unlike control mice. Despite this phenotype, bacterial burdens were not affected by loss of Vhl. In vitro studies demonstrated that transcriptional regulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand (RANKL) and its inhibitor osteoprotegerin (OPG) is altered in osteoblast-lineage cells with knockout of Vhl. After observing no impact on bacterial clearance with osteoblast-lineage conditional knockouts, a LysM-Cre model was used to generate Hif1aΔMyeloid and VhlΔMyeloid mouse models to explore the impact of myeloid cell HIF signaling. In both Hif1aΔMyeloid and VhlΔMyeloid models, bacterial clearance was not impacted. Moreover, minimal impacts on bone architecture were observed. Thus, skeletal HIF signaling was not found to impact bacterial clearance in our mouse model of post-traumatic osteomyelitis, but Vhl deletion in the osteoblast lineage was found to limit infection-mediated trabecular bone loss, possibly via altered regulation of RANKL-OPG gene transcription.

Phosphatidylinositol-4-kinase 2α enhances phagosomal TLR signaling and MHC class II presentation in mouse dendritic cells

Toll like receptor (TLR) recruitment to bacteria-containing phagosomes and subsequent TLR signaling in dendritic cells (DCs) are essential to initiate anti-microbial immune responses, but the mechanisms underlying TLR trafficking to phagosomes are poorly characterized. We previously showed that the endosomal adaptor protein-3 (AP-3) optimizes TLR4 and MyD88 recruitment to phagosomes and favors phagosomal TLR signaling to proinflammatory cytokine secretion, membrane tubulation and MHC-II presentation. However, AP-3 functions indirectly in these activities. We hypothesized that this reflects AP-3’s role in endolysosomal protein sorting, and that defining direct targets of AP-3 sorting will elucidate new membrane pathways controlling TLR signaling and anti-bacterial immune responses. We have identified that one such AP-3 effector is phosphatidylinositol (PI)-4-kinase 2α (PI4K2α), which generates PI4P, a lipid reported to engage the TLR adaptor TIRAP to recruit MyD88 and TIRAP to membranes. We show that PI4K2α is recruited to nascent LPS-bead phagosomes in wild-type (WT) bone marrow-derived (BM) DCs and that its recruitment is reduced in AP-3-deficient pearl BMDCs, both by immunofluorescence microscopy and immunoblotting of isolated phagosomes. Moreover, both PI4K2α-GFP and PI4P co-localize with LPS/OVA-texas red bead phagosomes 2 hours after uptake in WT DCs and on phagotubules. The formation of tubules from phagosomes in BMDCs is impaired upon knock-down of PI4K2α but not PI4K2β. This correlates with reduced MHC-II presentation of particulate Ealpha antigen and impaired T cell activation. Additionally, after LPS priming, IL-1β secretion induced by multiple stimuli is reduced in BMDCs by PI4K2α knock-down compared to controls, suggesting that PI4K2α is necessary for optimal inflammasome activity, likely by impacting either priming (via TLR signaling) or NLR stimulation (by increasing membrane surface via tubulation). In summary, PI4K2α is a novel regulator of phagosomal signaling required for TLR recruitment to phagosomes and downstream signaling pathways in DCs.

Tango of B cells with T cells in the making of secretory antibodies to gut bacteria.

Polymeric IgA and IgM are transported across the epithelial barrier from plasma cells in the lamina propria to exert a function in the gut lumen as secretory antibodies. Many secretory antibodies are reactive with the gut bacteria, and mounting evidence suggests that these antibodies are important for the host to control gut bacterial communities. However, we have incomplete knowledge of how bacteria-reactive secretory antibodies are formed. Antibodies from gut plasma cells often show bacterial cross-species reactivity, putting the degree of specificity behind anti-bacterial antibody responses into question. Such cross-species reactive antibodies frequently recognize non-genome-encoded membrane glycan structures. On the other hand, the T cell epitopes are peptides encoded in the bacterial genomes, thereby allowing a higher degree of predictable specificity on the T cell side of anti-bacterial immune responses. In this Perspective, we argue that the production of bacteria-reactive secretory antibodies is mainly controlled by the antigen specificity of T cells, which provide help to B cells. To be able to harness this system (for instance, for manipulation with vaccines), we need to obtain insight into the bacterial epitopes recognized by T cells in addition to characterizing the reactivity of the antibodies.

CD44a functions as a regulator of p53 signaling, apoptosis and autophagy in the antibacterial immune response

The cell adhesion molecule CD44 has been implicated in diverse biological functions including the pathological responses to infections and inflammatory diseases. The variable forms of CD44 contribute to functional variations, which are not yet defined in teleost. Here, we show that zebrafish CD44a plays a protective role in the host defense against Edwardsiella piscicida infection. Zebrafish CD44a deficiency inhibits cell growth and proliferation, impairs cell growth and death pathways, and regulates the expression levels of many genes involved in p53 signaling, apoptosis and autophagy. In addition, CD44a gene disruption in zebrafish leads to inhibition of apoptosis and induction of autophagy, with the increased susceptibility to E. piscicida infection. Furthermore, we show that zebrafish CD44a variants including CD44a_tv1 and CD44a_tv2 promote the translocation of p53 from the nucleus to the cytoplasm and interact with p53 in the cytoplasm. Mechanistically, zebrafish CD44a_tv1 mediates the beneficial effect for larvae survival infected with E. piscicida is depending on the CASP8-mediated apoptosis. However, the antibacterial effect of zebrafish CD44a_tv2 depends on the cytoplasmic p53-mediated inhibition of autophagy. Collectively, our results identify that different mechanisms regulate CD44a variants-mediated antibacterial responses.

The role of ficolin as a pattern recognition receptor in antibacterial immunity in Eriocheir sinensis

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named EsFicolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. EsFicolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant EsFicolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant EsFicolin could effectively remove bacteria and showed direct antibacterial activity. EsFicolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that EsFicolin plays an important role in the crab antibacterial immune response.