Muramyl dipeptide alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling.

Wnt signaling is a positive regulator of bone formation through the induction of osteoblast differentiation and down-regulation of osteoclast differentiation. We previously reported that muramyl dipeptide (MDP) increases bone volume by increasing osteoblast activity and attenuating osteoclast activity in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoporotic model mice. In this study, we investigated whether MDP could alleviate post-menopausal osteoporosis through Wnt signaling regulation in an ovariectomy (OVX)-induced mouse osteoporosis model. MDP-administered OVX mice exhibited higher bone volume and bone mineral density than mice of the control group. MDP significantly increased P1NP in the serum of OVX mice, implying increased bone formation. The expression of pGSK3ß and ß-catenin in the distal femur of OVX mice was lower than that in the distal femur of sham-operated mice. Yet, the expression of pGSK3ß and ß-catenin was increased in MDP-administered OVX mice compared with OVX mice. In addition, MDP increased the expression and transcriptional activity of ß-catenin in osteoblasts. MDP inhibited the proteasomal degradation of ß-catenin via the down-regulation of its ubiquitination by GSK3ß inactivation. When osteoblasts were pretreated with Wnt signaling inhibitors, DKK1 or IWP-2, the induction of pAKT, pGSK3ß, and ß-catenin was not observed. In addition, nucleotide oligomerization domain-containing protein 2-deficient osteoblasts were not sensitive to MDP. MDP-administered OVX mice exhibited fewer tartrate-resistant acid phosphatase (TRAP)-positive cells than did OVX mice, attributed to a decrease in the RANKL/OPG ratio. In conclusion, MDP alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling and could be an effective therapeutic for the treatment of post-menopausal bone loss. © 2023 The Pathological Society of Great Britain and Ireland.

Lipoteichoic acid inhibits osteoclast differentiation and bone resorption via interruption of gelsolin-actin dissociation.

Bone resorption can be caused by excessive differentiation and/or activation of bone-resorbing osteoclasts. While microbe-associated molecular patterns can influence the differentiation and activation of bone cells, little is known about the role of lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, in the regulation of bone metabolism. In this study, we investigated the effect of LTA on bone metabolism using wild-type Staphylococcus aureus and the LTA-deficient mutant strain. LTA-deficient S. aureus induced higher bone loss and osteoclast differentiation than wild-type S. aureus. LTA isolated from S. aureus (SaLTA) inhibited osteoclast differentiation from committed osteoclast precursors in the presence of various osteoclastogenic factors by downregulating the expression of NFATc1. Remarkably, SaLTA attenuated the osteoclast differentiation from committed osteoclast precursors of TLR2-/- or MyD88-/- mice and from the committed osteoclast precursors transfected with paired immunoglobulin-like receptor B-targeting siRNA. SaLTA directly interacted with gelsolin, interrupting the gelsolin-actin dissociation which is a critical process for osteoclastogenesis. Moreover, SaLTA suppressed the mRNA expression of dendritic cell-specific transmembrane protein, ATPase H+ transporting V0 subunit D2, and Integrin, which encode proteins involved in cell-cell fusion of osteoclasts. Notably, LTAs purified from probiotics, including Bacillus subtilis, Enterococcus faecalis, and Lactobacillus species, also suppressed Pam2CSK4- or RANKL-induced osteoclast differentiation. Taken together, these results suggest that LTAs have anti-resorptive activity through the inhibition of osteoclastogenesis by interfering with the gelsolin-actin dissociation and may be used as effective therapeutic agents for the prevention or treatment of inflammatory bone diseases.

Transparent Air Filters with Active Thermal Sterilization.

The worldwide proliferation of COVID-19 poses the urgent need for sterilizable and transparent air filters to inhibit virus transmission while retaining ease of communication. Here, we introduce copper nanowires to fabricate transparent and self-sterilizable air filters. Copper nanowire air filter (CNAF) allowed visible light penetration, thereby can exhibit facial expressions, helpful for better communication. CNAF effectively captured particulate matter (PM) by mechanical and electrostatic filtration mechanisms. The temperature of CNAF could be controlled by Joule-heating up to 100 °C with thermal stability. CNAF successfully inhibited the growth of E. coli because of the oligodynamic effect of copper. With heat sterilization, the antibacterial efficiency against G. anodireducens was greatly improved up to 99.3% within 10 min. CNAF showed high reusability with stable filtration efficiency and thermal antibacterial efficacy after five repeated uses. Our result suggests an alternative form of active antimicrobial air filter in preparation for the current and future pandemic situations.

Interleukin-10 and its receptors at the maternal-conceptus interface: expression, regulation, and implication for T helper 2 cytokine predominance and maternal immune tolerance in the pig, a true epitheliochorial placentation species†.

The appropriate balance between pro-inflammatory and anti-inflammatory cytokines is important for the maternal immune tolerance during pregnancy in mammals. Among the various cytokines, interleukin (IL)-10 (IL10) plays an essential role in anti-inflammatory responses, while IL12 is involved in pro-inflammatory responses during pregnancy. However, the roles of IL10 and IL12 in the endometrium during pregnancy have not been studied in pigs. Thus, we investigated the expression of IL10, IL12 (IL12A and IL12B), and their receptors (IL10RA, IL10RB, IL12RB1, and IL12RB2) at the maternal-conceptus interface. IL10, IL12, and their receptors were expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy stage-specific manner. During pregnancy, IL10 expression increased on Day 15, whereas the expression of IL12A and IL12B decreased after the implantation period. IL10 protein was localized to luminal epithelial (LE), stromal cells, and macrophages; IL10RA protein to LE, endothelial, stromal, and T cells; and IL10RB mRNA to LE cells in the endometrium. IL10 and IL10RA proteins and IL10RB mRNA were also localized to chorionic epithelial (CE) cells. In endometrial explants, the expression of IL10RA and IL10RB was induced by estradiol-17ß, IL-1ß, and/or interferon-γ. Heme oxygenase 1, an IL10-inducible factor, was expressed in the endometrium with the highest levels on Day 30 of pregnancy and was localized to LE and CE cells. These results in pigs suggest that conceptus-derived signals change the endometrial immune environment by regulating the expression of IL10 and IL10 receptors at the maternal-conceptus interface and that IL10 may provide anti-inflammatory conditions for the maternal immune tolerance.

Side branching and luminal lineage commitment by ID2 in developing mammary glands.

Mammary glands develop through primary ductal elongation and side branching to maximize the spatial area. Although primary ducts are generated by bifurcation of terminal end buds, the mechanism through which side branching occurs is still largely unclear. Here, we show that inhibitor of DNA-binding 2 (ID2) drives side branch formation through the differentiation of K6+ bipotent progenitor cells (BPs) into CD61+ luminal progenitor cells (LPs). Id2-null mice had side-branching defects, along with developmental blockage of the differentiation of K6+ BPs into CD61+ LPs. Notably, CD61+ LPs were found in budding and side branches, but not in terminal end buds. Hormone reconstitution studies using ovariectomized MMTV-hemagglutinin-nuclear localized sequence-tagged Id2 transgenic mice revealed that ID2 is a key mediator of progesterone, which drives luminal lineage differentiation and side branching. Our results suggest that CD61 is a marker of side branches and that ID2 regulates side branch formation by inducing luminal lineage commitment from K6+ BPs to CD61+ LPs.

Regulation of Bone Cell Differentiation and Activation by Microbe-Associated Molecular Patterns.

Gut microbiota has emerged as an important regulator of bone homeostasis. In particular, the modulation of innate immunity and bone homeostasis is mediated through the interaction between microbe-associated molecular patterns (MAMPs) and the host pattern recognition receptors including Toll-like receptors and nucleotide-binding oligomerization domains. Pathogenic bacteria such as Porphyromonas gingivalis and Staphylococcus aureus tend to induce bone destruction and cause various inflammatory bone diseases including periodontal diseases, osteomyelitis, and septic arthritis. On the other hand, probiotic bacteria such as Lactobacillus and Bifidobacterium species can prevent bone loss. In addition, bacterial metabolites and various secretory molecules such as short chain fatty acids and cyclic nucleotides can also affect bone homeostasis. This review focuses on the regulation of osteoclast and osteoblast by MAMPs including cell wall components and secretory microbial molecules under in vitro and in vivo conditions. MAMPs could be used as potential molecular targets for treating bone-related diseases such as osteoporosis and periodontal diseases.

Uterine epithelial expression of the tumor necrosis factor superfamily: a strategy for immune privilege during pregnancy in a true epitheliochorial placentation species.

The maternal immune system tolerates semi-allogeneic placental tissues during pregnancy. Fas ligand (FASLG) and tumor necrosis factor superfamily 10 (TNFSF10) are known to be components of maternal immune tolerance in humans and mice. However, the role of FASLG and TNFSF10 in the tolerance process has not been studied in pigs, which form a true epitheliochorial type placenta. Thus, the present study examined the expression and function of FASLG and TNFSF10 and their receptors at the maternal-conceptus interface in pigs. The endometrium and conceptus tissues expressed FASLG and TNFSF10 and their receptor mRNAs during pregnancy in a stage-specific manner. During pregnancy, FASLG and TNFSF10 proteins were localized predominantly to endometrial luminal epithelial cells with strong signals on Day 30 to term and on Day 15, respectively, and receptors for TNFSF10 were localized to some stromal cells. Interferon-γ (IFNG) increased the expression of TNFSF10 and FAS in endometrial tissues. Co-culture of porcine endometrial epithelial cells over-expressing TNFSF10 with peripheral blood mononuclear cells yielded increased apoptotic cell death of lymphocytes and myeloid cells. In addition, many apoptotic T cells were found in the endometrium on Day 15 of pregnancy. The present study demonstrated that FASLG and TNFSF10 were expressed at the maternal-conceptus interface and conceptus-derived IFNG increased endometrial epithelial TNFSF10, which, in turn, induced apoptotic cell death of immune cells. These results suggest that endometrial epithelial FASLG and TNFSF10 may be critical for the formation of micro-environmental immune privilege at the maternal-conceptus interface for the establishment and maintenance of pregnancy in pigs.

Characterization of splenic MRC1hiMHCIIlo and MRC1loMHCIIhi cells from the monocyte/macrophage lineage of White Leghorn chickens.

Monocytes/macrophages, which are found in a variety of organs, maintain tissue homeostasis at a steady state and act as the first line of defence during pathogen-induced inflammation in the host. Most monocyte/macrophage lineage studies in chickens have been largely performed using cell lines, while few studies using primary cells have been conducted. In the present study, the phenotypic and functional characteristics of splenic monocyte/macrophage lineage cells during steady state and inflammatory conditions were examined. Splenic monocyte/macrophage lineage cells could be identified as MRC1loMHCIIhi and MRC1hiMHCIIlo cells based on their surface expression of MRC1 and MHCII. In the steady state, MRC1loMHCIIhi cells were more frequently found among MRC1+ cells. MRC1loMHCIIhi cells expressed a higher number of antigen-presenting molecules (MHCII, MHCI, and CD80) than MRC1hiMHCIIlo cells. In contrast, MRC1hiMHCIIlo cells showed better phagocytic and CCR5-dependent migratory properties than MRC1loMHCIIhi cells. Furthermore, MRC1hiMHCIIlo cells infiltrated the spleen in vivo and then became MRC1loMHCIIhi cells. During lipopolysaccharide (LPS)-induced inflammatory conditions that were produced via intraperitoneal (i.p.) injection, the proportion and absolute number of MRC1hiMHCIIlo cells were increased in the spleen. Uniquely, inflammation induced the downregulation of MHCII expression in MRC1hiMHCIIlo cells. The major source of inflammatory cytokines (IL-1ß, IL-6, and IL-12) was MRC1loMHCIIhi cells. Furthermore, MRC1hiMHCIIlo cells showed greater bactericidal activity than MRC1loMHCIIhi cells during LPS-induced inflammation. Collectively, these results suggest that two subsets of monocyte/macrophage lineage cells exist in the chicken spleen that have functional differences.

Bacillus subtilis spores as adjuvants against avian influenza H9N2 induce antigen-specific antibody and T cell responses in White Leghorn chickens.

Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1ß and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4+ and CD8+ T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4+ and CD8+ T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.

Replication of Vibrio cholerae classical CTX phage.

The toxigenic classical and El Tor biotype Vibrio cholerae serogroup O1 strains are generated by lysogenization of host-type-specific cholera toxin phages (CTX phages). Experimental evidence of the replication and transmission of an El Tor biotype-specific CTX phage, CTX-1, has explained the evolution of V. cholerae El Tor biotype strains. The generation of classical biotype strains has not been demonstrated in the laboratory, and the classical biotype-specific CTX phage, CTX-cla, is considered to be defective with regard to replication. However, the identification of atypical El Tor strains that contain CTX-cla-like phage, CTX-2, indicates that CTX-cla and CTX-2 replicate and can be transmitted to V. cholerae strains. The replication of CTX-cla and CTX-2 phages and the transduction of El Tor biotype strains by various CTX phages under laboratory conditions are demonstrated in this report. We have established a plasmid-based CTX phage replication system that supports the replication of CTX-1, CTX-cla, CTX-2, and CTX-O139. The replication of CTX-2 from the tandem repeat of lysogenic CTX-2 in Wave 2 El Tor strains is also presented. El Tor biotype strains can be transduced by CTX phages in vitro by introducing a point mutation in toxT, the transcriptional activator of the tcp (toxin coregulated pilus) gene cluster and the cholera toxin gene. This mutation also increases the expression of cholera toxin in El Tor strains in a sample single-phase culture. Our results thus constitute experimental evidence of the genetic mechanism of the evolution of V. cholerae.

Modulation of macrophage subtypes by IRF5 determines osteoclastogenic potential.

Bone-resorbing osteoclasts are differentiated from macrophages (MΦ) by M-CSF and RANKL. MΦ can be mainly classified into M1 and M2 MΦ, which are proinflammatory and anti-inflammatory, respectively, but little is known about their osteoclastogenic potential. Here, we investigated the osteoclastogenic potential of MΦ subtypes. When the two MΦ subtypes were differentiated into osteoclasts using M-CSF and RANKL, M2 MΦ more potently differentiated into osteoclasts than M1 MΦ. M2 MΦ generated with IL-4 or IL-10 also showed enhanced osteoclast differentiation compared with M1 MΦ induced by IFN-γ and lipopolysaccharide. In addition, robust bone-resorptive capacity and giant actin rings, which are features of mature osteoclasts, were observed in M2, but not M1 MΦ, under the osteoclast differentiation condition. Osteoclast differentiation was significantly increased in CD206+ M2 MΦ but not in CD86+ M1 MΦ. Compared with M1 MΦ, c-Fms and RANK were highly expressed in M2 MΦ. Enhanced osteoclastogenesis of M2 MΦ was mediated through sustained ERK activation, followed by efficient c-Fos and NFATc1 induction. Notably, the osteoclastogenic potential of M1 MΦ converted into M2 MΦ by exposure to M-CSF was higher than that of M2 MΦ converted into M1 MΦ by exposure to GM-CSF. Silencing IRF5, which is responsible for M1 MΦ polarization, increased osteoclast differentiation by enhancing c-Fms expression and activation of ERK, c-Fos, CREB, and NFATc1, which was inhibited by overexpression of IRF5. Collectively, M2 MΦ are suggested to be more efficient osteoclast precursors than M1 MΦ because of the attenuated expression of IRF5.

Streptococcus gordonii induces bone resorption by increasing osteoclast differentiation and reducing osteoblast differentiation.

Streptococcus gordonii is commonly found in the periapical endodontic lesions of patients with apical periodontitis, a condition characterized by inflammation and periapical bone loss. Since bone metabolism is controlled by osteoclastic bone resorption and osteoblastic bone formation, we investigated the effects of S. gordonii on the differentiation and function of osteoclasts and osteoblasts. For the determination of bone resorption activity in vivo, collagen sheets soaked with heat-killed S. gordonii were implanted on mouse calvaria, and the calvarial bones were scanned by micro-computed tomography. Mouse bone marrow-derived macrophages (BMMs) were stimulated with M-CSF and RANKL for 2 days and then differentiated into osteoclasts in the presence or absence of heat-killed S. gordonii. Tartrate-resistant acid phosphatase staining was performed to determine osteoclast differentiation. Primary osteoblast precursors were differentiated into osteoblasts with ascorbic acid and ß-glycerophosphate in the presence or absence of heat-killed S. gordonii. Alkaline phosphatase staining and alizarin red S staining were conducted to determine osteoblast differentiation. Western blotting was performed to examine the expression of transcription factors including c-Fos, NFATc1, and Runx2. Heat-killed S. gordonii induced bone destruction in a mouse calvarial implantation model. The differentiation of RANKL-primed BMMs into osteoclasts was enhanced in the presence of heat-killed S. gordonii. Heat-killed S. gordonii increased the expression of c-Fos and NFATc1, which are essential transcription factors for osteoclast differentiation. On the other hand, heat-killed S. gordonii inhibited osteoblast differentiation and reduced the expression of Runx2, an essential transcription factor for osteoblast differentiation. S. gordonii exerts bone resorptive activity by increasing osteoclast differentiation and reducing osteoblast differentiation, which may be involved in periapical bone resorption.

Human CD141+ dendritic cells generated from adult peripheral blood monocytes.

Human CD141+ dendritic cells (DCs), specialized for cross-presentation, have been extensively studied in the development of DC-based therapy against cancer. A series of attempts was made to generate CD141+ DCs from cord blood CD34+ hematopoietic progenitors to overcome the practical limitation of in vivo rareness. In the present study, we identified a culture system that generates high CD141+ DCs. After culture of CD14+ monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 8 days, CD141 was detected on cells that adhered to the bottom of the culture plate. The attached cells exhibited typical features of immature monocyte-derived DCs (moDCs), except for higher CD86 expression, more dendrites and higher granularity compared with those that did not attach. With 3 additional days of culture, increased CD141 expression on the cells was retained along with adhesion ability and partial expression of CLEC9A, a c-type lectin receptor. Furthermore, the cells exhibited effective uptake of dead cells. Interestingly, the attached moDCs differently responded to polyinosinic:polycytidylic acid (poly I:C) stimulation as well as a mixed lymphocyte reaction. Collectively, our findings show that human CD141+ DCs can be sufficiently generated from peripheral blood CD14+ monocytes, potentiating further investigation into generation of higher yields of cross-priming human DCs in vitro.

Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

Cysteine-X-cysteine (CXC) motif chemokine ligand 12 (CXCL12) and its receptor, CXC chemokine receptor type 4 (CXCR4), are involved in regulating the proliferation, migration, and survival of trophoblast cells and the maternal immune response in humans and mice. The present study examined the expression, regulation, and function of CXCL12 and CXCR4 at the maternal-conceptus interface during pregnancy in pigs. The endometrium expressed CXCL12 and CXCR4 mRNAs with the greatest CXCL12 abundance on Day 15 of pregnancy. CXCL12 protein was localized mainly in endometrial epithelial cells, while CXCR4 protein was localized in subepithelial stromal cells, vascular endothelial cells, and immune cells in blood vessels in the endometrium during the estrous cycle and pregnancy. CXCL12 protein was detected in uterine flushing on Day 15 of pregnancy. The conceptus during early pregnancy and chorioallantoic tissues during mid-to-late pregnancy expressed CXCL12 and CXCR4. Interferon-γ increased the abundance of CXCL12, but not CXCR4 mRNA in endometrial explants. Recombinant CXCL12 (rCXCL12) protein dose-dependently increased migration of cultured porcine trophectoderm cells and peripheral blood mononuclear cells (PBMCs). Furthermore, rCXCL12 caused migration of T cells, but not natural killer cells, in PBMCs. This study revealed that interferon-γ-induced CXCL12 and its receptor, CXCR4, were expressed at the maternal-conceptus interface and increased the migration of trophectoderm cells and T cells at the time of implantation in pigs. These results suggest that CXCL12 may be critical for the establishment of pregnancy by regulating trophoblast migration and T cell recruitment into the endometrium during the implantation period in pigs.

Staphylococcus aureus lipoproteins augment inflammatory responses in poly I:C-primed macrophages.

Secondary bacterial infection contributes to severe inflammation following viral infection. Among foodborne pathogenic bacteria, Staphylococcus aureus is known to exacerbate severe inflammatory responses after infection with single-stranded RNA viruses such as influenza viruses. However, it has not been determined if S. aureus infection enhances inflammatory responses after infection with RNA enteric viruses, including rotavirus, which is a double-stranded RNA virus. We therefore investigated the molecular mechanisms by which a cell wall component of S. aureus enhanced inflammatory responses during enteric viral infection using poly I:C-primed macrophages, which is a well-established model for double-stranded RNA virus infection. S. aureus lipoproteins enhanced IL-6 as well as TNF-α production in poly I:C-primed macrophages. Pam2CSK4, a mimic of Gram-positive bacterial lipoproteins and S. aureus lipoproteins, also significantly enhanced IL-6 production in poly I:C-primed macrophages. While IFN-ß expression was increased in poly I:C-primed macrophages treated with Pam2CSK4 or S. aureus lipoproteins, the level of IL-6 enhancement in poly I:C-primed macrophages was decreased in the presence of anti-IFN-α/ß receptor antibody, suggesting that IFN-ß plays an important role in enhanced IL-6 production. Phosphatidylinositol-3-kinase, Akt, ERK and NF-κB were also involved in the enhanced IL-6 production. Collectively, these results suggest that S. aureus lipoproteins induce excessive inflammatory responses in the presence of poly I:C.

Gamma-irradiation-killed Streptococcus pneumoniae potently induces the expression of IL-6 and IL-8 in human bronchial epithelial cells.

Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been developed and its immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immunity, in the present study, we investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cell-line, BEAS-2B, by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP). r-SP potently induced interleukin (IL)-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner, whereas h-SP and f-SP poorly induced them. Of note, the mRNA levels of IL-6 and IL-8 were approximately two-fold higher when cells were stimulated with 3 × 107 CFU/ml of r-SP for 3 h, while the protein levels of IL-6 and IL-8 were approximately five-fold higher after stimulation with 3 × 107 CFU/ml of r-SP for 24 h. Furthermore, r-SP exhibited potent activation of Toll-like receptor 2 compared with h-SP or f-SP. The expression of IL-6 and IL-8 induced by r-SP was mediated through the activation of mitogen-activated protein kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the aforementioned activities. Taken together, we suggest that r-SP stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8, which might influence the induction of adaptive immune responses.

Analysis of cysteine-X-cysteine motif chemokine ligands 9, 10, and 11, their receptor CXCR3, and their possible role on the recruitment of immune cells at the maternal-conceptus interface in pigs.

Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period in pigs.

Effects of ambient temperature and dietary glycerol addition on growth performance, blood parameters and immune cell populations of Korean cattle steers.

OBJECTIVE: This study was performed to evaluate whether ambient temperature and dietary glycerol addition affect growth performance, and blood metabolic and immunological parameters, in beef cattle. METHODS: Twenty Korean cattle steers (405.1±7.11 kg of body weight [BW], 14.2±0.15 months of age) were divided into a conventional control diet group (n = 10) and a 2% glycerol- added group (n = 10). Steers were fed 1.6% BW of a concentrate diet and 0.75% BW of a timothy hay diet for 8 weeks (4 weeks from July 28th to August 26th and 4 weeks from August 27th to September 26th). Blood was collected four times on July 28th, August 11th, August 27th, and September 26th. RESULTS: The maximum indoor ambient temperature-humidity index in August (75.8) was higher (p<0.001) than that in September (70.0), and in August was within the mild heat stress (HS) category range previously reported for dairy cattle. The average daily gain (ADG; p = 0.03) and feed efficiency (p<0.001) were higher in hotter August than in September. Glycerol addition did not affect ADG and feed efficiency. Neither month nor glycerol addition affected blood concentrations of cortisol, triglyceride, or non-esterified fatty acid. Blood concentrations of cholesterol, low-density lipoprotein, high-density lipoprotein, glucose, and albumin were lower (p<0.05) on August 27th than on September 26 th, and blood phosphorus, calcium and magnesium concentrations were also lower on August 27th than on September 27th. Glycerol addition did not affect these blood parameters. Percentages of CD4+ T cells and CD8+ T cells were higher (p<0.05) on July 28th than on August 27th and September 26th. The blood CD8+ T cell population was lower in the glycerol supplemented-group compared to the control group on July 28th and August 27th. CONCLUSION: Korean cattle may not be significantly affected by mild HS, considering that growth performance of cattle was better in hotter conditions, although some changes in blood metabolic and mineral parameters were observed.

Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.

Typhoid fever, mainly caused by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri. Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 (P < 0.001). Taken together, the semi-automated SBA might be efficient, accurate, sensitive, and specific enough to measure functional antibody titers against S. Typhi in sera from human subjects immunized with typhoid vaccines.

Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.