MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus

MyD88 is an important signaling adaptor for both TLR and IL-1R family members. Here, we evaluated the role of TLR2/MyD88 and IL-1R/MyD88 signaling in host defense against S. aureus by using a cutaneous infection model in conjunction with bioluminescent bacteria. We found that lesions of S. aureus-infected MyD88- and IL-1R-deficient mice were substantially larger with higher bacterial counts compared with wild-type mice. In contrast, TLR2-deficient mice had lesions that were only moderately larger with minimally higher bacterial counts. In addition, MyD88- and IL-1R- but not TLR2-deficient mice had severely decreased recruitment of neutrophils to the site of infection. This neutrophil recruitment was not dependent upon IL-1R/MyD88 signaling by recruited bone marrow-derived cells, suggesting that resident skin cells utilize IL-1R/MyD88 signaling to promote neutrophil recruitment.     

Bone Marrow Stromal Cells (BMSCs) in Bone Engineering: Limitations and Recent Advances

Bone marrow stromal cells (BMSCs) have been isolated for the first time by Friedenstein et al. and since then have been considered the progenitor cells for the skeletal tissues. Indeed BMSCs are clonogenic, fibroblastic in shape, and can differentiate along multiple lineages such as osteoblasts, chondrocytes, adipocytes, and hematopoiesis-supportive stroma. When implanted in vivo on a three-dimensional bioceramic scaffold into immunocompromised mice, BMSCs form bone and hematopoiesis-supportive stroma. The ease of harvest from a donor bone marrow together with the ability to form bone in vivo make BMSCs ideal for clinical applications. Thus, ex vivo expanded BMSCs have been employed, first in large animal models, then in human clinical trials, to repair large bone segmental defects. Further investigation of the expanded BMSC population led to the observation that in vitro expansion appears a limiting passage: cells tend to senesce and lose their multidifferentiation potential with time in culture. To overcome these limitations, two approaches have been proposed: (1) identification of the appropriate culture conditions to prevent senescence by possibly selecting a subpopulation with stem cell characteristics, and (2) engineering of the cells by transfection with the telomerase gene to prevent cells from telomere shortening and consequent aging.     

Human Stromal (Mesenchymal) Stem Cells from Bone Marrow, Adipose Tissue and Skin Exhibit Differences in Molecular Phenotype and Differentiation Potential

Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34^?, CD45^?, CD14^?, CD31^?, HLA-DR^?, CD13⁺, CD29⁺, CD44⁺, CD73⁺, CD90⁺,and CD105⁺. When exposed to in vitro differentiation, hATSCs, hASSCs and hNSSCs exhibited quantitative differences in their ability to differentiate into adipocytes and to osteoblastic cells. Using a microarray-based approach we have unveiled a common MSC molecular signature composed of 33 CD markers including known MSC markers and several novel markers e.g. CD165, CD276, and CD82. However, significant differences in the molecular phenotype between these different stromal cell populations were observed suggesting ontological and functional differences. In conclusion, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols.     

兔BMSCs体外培养及其向成骨细胞分化的实验研究

目的　探讨培养兔骨髓基质干细胞向成骨细胞的分化,为骨组织工程研究提供种子细胞。方法　取 2月龄新西兰大耳白兔,麻醉后取骨髓,直接进行原代培养,传代后观察其生长特性,绘制生长曲线并加诱导液使其 向成骨细胞方向分化,并分别用钙钴法检测碱性磷酸酶,茜素红染色检测钙结节,免疫组化染色检测Ⅰ型胶原,透 射电镜观察胞质中钙质成分。结果　原代培养中出现大量细胞克隆,传代后细胞呈旋涡状密集生长,加入诱导液 后细胞形态发生改变并向成骨细胞分化,胞质内见有呈黑色的碱性磷酸酶颗粒和Ⅰ型胶原反应产物,并见有多个 细胞形成的钙化结节,电镜下观察到胞质中含有许多基质小泡,几天内成骨细胞数可达1×106/L。结论　培养兔 骨髓基质干可向成骨细胞方向分化,作为骨组织工程的种子细胞。     

Schistosoma mansoni egg antigen-mediated modulation of Toll-like receptor (TLR)-induced activation occurs independently of TLR2, TLR4, and MyD88

Unlike most pathogens, helminth parasites and their products induce strong Th2 responses, and dendritic cells (DCs) and macrophages exposed to helminth antigens generally fail to produce interleukin-12. Rather, it has been shown that helminth products such as soluble egg antigens (SEA; a soluble extract from Schistosoma mansoni eggs) inhibit the activation of DCs in response to classical Toll-like receptor (TLR) ligands such as lipopolysaccharide or CpG. Nevertheless, recent work has suggested that TLR4 and/or TLR2 plays an important role in the recognition of helminth products by DCs and macrophages and in the development of Th2 responses. Using DCs derived from TLR4^−/−, TLR2^−/−, or MyD88^−/− mice, we have demonstrated that the ability of SEA to modulate DC activation is MyD88 independent and requires neither TLR4 nor TLR2. Moreover, TLR2 and TLR4 are not required for SEA-pulsed DCs to induce Th2 responses in naïve mice.Helminth parasites, which colonize organ systems as diverse as the lymphatics, gastrointestinal tract, and vasculature, have evolved multiple immunomodulatory mechanisms to evade host immune responses (20). A delicate balance is required in these chronic infections to establish parasite survival without eliciting lethal immunopathology. This balance is illustrated in schistosomiasis, which is caused by the trematode Schistosoma mansoni and causes chronic morbidity in more than 200 million people (24). Following infection, worms migrate to the portal vasculature, where they mature and pair. This early phase of infection is characterized by a Th1 response. After worm pairing, females lay eggs that cross the intestinal barrier to be excreted in the feces. However, some eggs become lodged in the intestinal wall and liver sinusoids, where soluble egg antigens (SEA) induce a polarized Th2 response (23). The Th2 response correlates with the downmodulation of the initial proinflammatory Th1 response to migrating immature worms and results in granuloma formation. Failure to switch to a Th2 response leads to hepatotoxic liver disease and host death (3, 6, 11).The mechanism by which host innate immune cells recognize SEA remains unclear. Pathogens such as bacteria, viruses, and intracellular parasites express conserved molecular signatures that are shared within classes of pathogens and their free-living relatives. These are recognized by highly conserved pattern recognition receptors (PRRs) expressed by innate defense cells, including dendritic cells (DCs) and macrophages. PRRs include C-type lectins and Toll-like receptors (TLRs) (10, 32). TLRs are the most well-described PRRs, and DC activation by TLR ligation is considered to play a major role in the coordination of innate and adaptive immune responses during infection (22). Typically, the ligation of TLRs initiates a proinflammatory program, which promotes innate defense mechanisms and adaptive Th1 or Th17 immune responses to the invasive pathogens (22). There is evidence, however, that lipopolysaccharide (LPS) activation of TLR4 can induce DCs to support the development of Th2 responses (5, 15).Emerging data have demonstrated that phospholipids or glycoproteins unique to extracellular helminths ligate TLRs to induce an anti-inflammatory and Th2-inducing antigen-presenting cell phenotype. A phosphorylcholine-containing glycoprotein, ES-62, from the nematode Acanthocheilonema viteae, has been shown to induce a polarized Th2 response and to work via TLR4 to modulate antigen-presenting cell activation by a variety of TLR ligands (7, 33). There is also evidence that S. mansoni products can stimulate antigen-presenting cells through TLRs. A lipid fraction from S. mansoni eggs containing lysophosphatidylserine has been shown, in a TLR2-dependent mechanism, to induce the activation of DCs that promote Th2 and regulatory T-cell development (28), and lacto-N-fucopentaose III (LNFPIII), a synthetic copy of a schistosome egg glycan, has been shown to promote Th2 differentiation by DCs via a TLR4-dependent pathway (26).Here, using gene-targeted mice, we demonstrate conclusively that the anti-inflammatory and Th2-inducing characteristics of SEA are MyD88 independent and require neither TLR2 nor TLR4.     

Induction of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Granulocyte Colony-Stimulating Factor (G-CSF) Expression in Bone Marrow and Fractionated Marrow Cell Populations by Interleukin 3 (IL-3): IL-3-Mediated Positive Feedback Mechanisms of Granulopoiesis

Abstract

Interleukin 3 (IL-3) induces proliferation and differentiation of mast cell progenitors in vitro, whereas it induces granulocytosis in vivo. In this paper, a positive feedback mechanism of granulopoiesis was studied in order to elucidate the granulocytosis induced by IL-3 in mouse. IL-3 induced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in total bone marrow cells and a marrow adherent cell population. In fractionated marrow cell populations, a different expression pattern of induction by IL-3 stimulation was observed; GM-CSF was expressed in macrophages and fraction 1 (P<1.061) and 2(1.061<P<1.074) of bone marrow cells fractionated by equilibrium density centrifugation, G-CSF was expressed in macrophages and fraction 2 and 3 (1.074<P<1.097), and interleukin 6 (IL-6) in macrophages and fraction 1 to 3. These results indicate a hierarchical regulation of cytokine production and the existence of a positive feedback mechanism in granulopoiesis. IL-6, induced by IL-3, stimulates stem cells into cycle and induces stem cells to respond to IL-3. The stem cells differentiate to granulocyte-macrophage colony-forming cells by the combined effect of IL-3 and IL-6. IL-3 also induces GM- and G-CSF expression which in turn makes granulocyte-macrophage colony-forming cells differentiate to granulocytes. These factors organize a cytokine network in granulopoiesis.     

In Vitro Mesenchymal Trilineage Differentiation and Extracellular Matrix Production by Adipose and Bone Marrow Derived Adult Equine Multipotent Stromal Cells on a Collagen Scaffold

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.     

GATA-1和GATA-2基因在再障和正常骨髓基质细胞中的表达

目的探讨转录因子GATA-1和GATA-2基因在再障和正常骨髓基质细胞中表达的情况.方法体外扩增培养骨髓基质细胞,运用RT-PCR-ELISA方法检测GATA-1和GATA-2基因的表达情况,并对其相对表达水平进行半定量比较.结果 GATA-1和GATA-2基因在正常和再障的骨髓基质细胞中均有一定的表达.再障的骨髓基质细胞中GATA-1的表达水平较正常低;而GATA-2的表达水平与正常相比无显著性差异.正常和再障的骨髓基质细胞中均以GATA-2的表达占优势.结论转录因子GATA-1和GATA-2基因可在正常和再障骨髓基质细胞中表达,并可能影响骨髓微环境的造血调控作用,对再障等血液疾病的发生发展起一定的作用.     

GATA-1和GATA-2基因在再障和正常骨髓基质细胞中的表达

目的 探讨转录因子GATA-1和GATA-2基因在再障和正常骨髓基质细胞中表达的情况。方法 体外扩增培养骨髓基质细胞，运用RT-PCR—ELISA方法检测GATA-1和GATA-2基因的表达情况，并对其相对表达水平进行半定量比较。结果 GATA-1和GATA-2基因在正常和再障的骨髓基质细胞中均有一定的表达。再障的骨髓基质细胞中GATA-1的表达水平较正常低；而GATA-2的表达水平与正常相比无显著性差异．正常和再障的骨髓基质细胞中均以GATA-2的表达占优势．结论 转录因子GATA-1和GATA-2基因可在正常和再障骨髓基质细胞中表达，并可能影响骨髓微环境的造血调控作用，对再障等血液疾病的发生发展起一定的作用。     

Interleukin-1 (IL-1) Signaling in Intestinal Stromal Cells Controls KC/CXCL1 Secretion,Which Correlates with Recruitment of IL-22-Secreting Neutrophils at Early Stages of Citrobacter rodentium Infection

Attaching and effacing pathogens, including enterohemorrhagic Escherichia coli in humans and Citrobacter rodentium in mice, raise serious public health concerns. Here we demonstrate that interleukin-1 receptor (IL-1R) signaling is indispensable for protection against C. rodentium infection in mice. Four days after infection with C. rodentium, there were significantly fewer neutrophils (CD11b⁺ Ly6C⁺ Ly6G⁺) in the colons of IL-1R^−/− mice than in wild-type mice. Levels of mRNA and protein of KC/CXCL1 were also significantly reduced in colon homogenates of infected IL-1R^−/− mice relative to wild-type mice. Of note, infiltrated CD11b⁺ Ly6C⁺ Ly6G⁺ neutrophils were the main source of IL-22 secretion after C. rodentium infection. Interestingly, intestinal stromal cells isolated from IL-1R^−/− mice secreted lower levels of KC/CXCL1 than stromal cells from wild-type mice during C. rodentium infection. Similar effects were found when mouse intestinal stromal cells and human nasal polyp stromal cells were treated with IL-1R antagonists (i.e., anakinra) in vitro. These results suggest that IL-1 signaling plays a pivotal role in activating mucosal stromal cells to secrete KC/CXCL1, which is essential for infiltration of IL-22-secreting neutrophils upon bacterial infection.     

Osteogenic Differentiation of Marrow Stromal Cells on Random and Aligned Electrospun Poly(l-lactide) Nanofibers

The fibrillar structure and sub-micron diameter of electrospun nanofibers can be used to reproduce the morphology and structure of the natural extracellular matrix (ECM). The objective of this work was to investigate the effect of fiber alignment on osteogenic differentiation of bone marrow stromal (BMS) cells. Random and aligned poly(l-lactide) (PLLA) nanofibers were produced by collecting the spun fibers on a stationary plate and a rotating wheel, respectively, as the ground electrode. Morphology and alignment of the BMS cells seeded on the fibers were characterized by SEM. The effect of fiber orientation on osteogenic differentiation of BMS cells was determined by measuring alkaline phosphatase (ALPase) activity, calcium content, and mRNA expression levels of osteogenic markers. There was a strong correlation between the fiber and cell distributions for the random (p = 0.16) and aligned (p = 0.81) fibers. Percent deviation from ideal randomness (PDIR) values indicated that cells seeded on the random fibers (PDIR = 6.5%) were likely to be distributed randomly in all directions while cells seeded on the aligned fibers (PDIR = 86%) were highly likely to be aligned with the direction of fibers. BMS cell seeded on random and aligned fibers had similar cell count and ALPase activity with incubation time, but the calcium content on aligned fibers was significantly higher after 21 days compared to that of random fibers (p = 0.003). Osteopontin (OP) and osteocalcin (OC) expression levels of BMS cells on fibers increased with incubation time. However, there was no difference between the expression levels of OP and OC on aligned vs. random fibers. The results indicate that BMS cells aligned in the direction of PLLA fibers to form long cell extensions, and fiber orientation affected the extent of mineralization, but it had no effect on cell proliferation or mRNA expression of osteogenic markers.     

Ligands of NOD2 (Muramyl Dipeptide) and TLR4 (LPS) in 24 h after Combined In Vivo Administration Produce a Synergistic Increase in the Content of Multipotent Stromal Cells in the Bone Marrow and Peritoneal Exudate of CBA Mice

Bulletin of Experimental Biology and Medicine - In 24 h after combined administration of ligands of NOD2 (muramyl dipeptide) and TLR4 (LPS) receptors to CBA mice, a synergistic increase (by 10...     

Serial Examination of Serum IL-8, IL-10 and IL-1Ra Levels is Significant in Neonatal Seizures Induced by Hypoxic-Ischaemic Encephalopathy(1)

We investigated changes in the levels of significant cytokines in relation to neonatal seizures, a pattern of cytokine concentrations serially and the severity of brain insult. The hypoxic–ischaemic encephalopathy‐induced seizure group consisted of 13 patients, and another 15 normal newborns were enrolled as a control group. All of the initial samples were obtained within the first 24 h of admission, and the second samples were obtained between 48 and 72 h in both groups. Only the third samples were taken in the seizure group on the 5th day. During neonatal seizures, the levels of most cytokines increased within 24 h, and, in particular, the levels of interleukin (IL)‐8 significantly increased (P < 0.05). After 48–72 h of seizure onset, the levels of most cytokines decreased, especially, IL‐1Ra; however, IL‐8 and IL‐10 remained increased (P < 0.05). During the prognosis, one patient who was diagnosed with quadriplegic cerebral palsy at 6 months of age presented extreme elevation of IL‐1beta, IL‐1Ra, IL‐6, IL‐8, IL‐10 and tumor necrosis factor‐alpha in the initial sample, reflecting the severity of brain damage. A significant increase in IL‐8 may serve as a biomarker for earlier detection of brain damage in neonatal seizure, if detected within 24 and 48–72 h of the seizure.     

Very Early Onset Inflammatory Bowel Disease Associated with Aberrant Trafficking of IL-10R1 and Cure by T Cell Replete Haploidentical Bone Marrow Transplantation

Purpose

Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT).

Methods

Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry.

Results

We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3′ splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient’s cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients.

Conclusions

Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.     

人甲状旁腺激素调节骨保护素及其配体基因表达的PKA、PKC信号传导通路研究

目的探讨PTH调节成骨细胞OPG/RANKL mRNA表达的信号传导通路。方法采用原代培养的胎鼠颅盖骨成骨细胞,在细胞培养5 d和6 d后,分别加入抑制PKC和PKA传导通路的Calphostin-C 500 nmol/L24 h和H-89 30μmol/L 4 h。在细胞培养5 d后,加入刺激PKC传导通路的PMA10-7mol/L24 h后,分别加入和不加入hPTH(1-34)100 ng/ml,继续培养2 h和6 h分别收集细胞,采用RT-PCR方法半定量检测OPG和RANKL基因的表达变化。结果PKA通路抑制剂H-89 30μmol/L能显著改变100 ng/ml hPTH(1-34)导致的OPG和RANKL基因的表达趋势,表现为上调OPG基因和下调RANKL基因(35%和59%)。而PKC通路激动剂和抑制剂对100 ng/ml hPTH(1-34)导致的OPG和RANKL基因的表达趋势无影响。结论hPTH持续刺激上调RANKL基因,下调OPG基因的表达,主要是通过PKA通路介导,在此过程中,PKC通路无作用。     

Simultaneous Administration of NOD-2 (MDP) and TLP-4 (LPS) Ligands to Bone Marrow Donors 24 h before Transplantation Increases the Content of Multipotent Stromal Cells (MSCs) in Bone Marrow Grafts in CBA Mice Compared to the Total Result of Their Isolated Administration

Bulletin of Experimental Biology and Medicine - In 3-month bone marrow transplants of CBA mice from bone marrow donors receiving single injections of TLR-4 ligand (LPS) or NOD-2 ligand (muramyl...