Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Electroacupuncture preconditioning attenuates acute myocardial ischemia injury through inhibiting NLRP3 inflammasome activation in mice.

AIMS: Electro-acupuncture pretreatment (EAP) plays a protective role in myocardial ischemia (MI) injury. However, the underlying mechanism remains unclear. A growing body of evidence suggests postinfarction inflammatory response directly affects the remodeling of ventricular function. The purpose of this study was to investigate whether EAP alleviates MI through NLRP3 inflammasome inhibition. MATERIALS AND METHODS: We constructed an AMI model by ligating the left anterior descending (LAD) coronary artery after 3 days of EAP with C57BL/6 mice. Echocardiography and TTC staining were employed to evaluate cardiac function and infarct size after 24 h of ischemia. HE staining and immunohistochemistry were employed to determine inflammatory level. Then, inflammasome activation was detected by western blotting, and macrophage polarization and neutrophil infiltration were observed by flow cytometry. KEY FINDINGS: Our preliminary findings showed that EAP reduced the infarct area and increased fractional shortening (FS) and ejection fraction (EF) and decreased the degree of inflammation after AMI injury. Meanwhile, EAP inhibited the expression of NLRP3, cleaved caspase-1 and IL-1ß in ischemia myocardial tissue, companied by inhibiting the expression of F4/80+, CD11b+, CD206low macrophages and activated M2 macrophage, and decreasing Ly-6G+CD11b+ neutrophils in ischemia myocardial and spleen tissue. SIGNIFICANCE: EAP inhibits the activation of NLRP3 inflammasome, promotes M2 polarization of macrophages and reduces the recruitment of neutrophils in damaged myocardium, thereby decreases the infarct size and improves the cardiac function.

[Study on anti-aging effect of ginsenoside Rg1 in serial transplantation of hematopoietic stem cells and progenitor cells].

OBJECTIVE: To investigate the anti-aging effect of ginsenoside R1 in serial transplantation of hematopoietic stem cells and progenitor cells. METHOD: HSC/HPC aging model in vivo was established through the Sca-1 (+) HSC/HPC serial transplantation of male donor mice that had been separated and purified by the magnetic-activated cell sorting method. The female recipient mice that had been radiated with lethal dose of 60Co gamma ray were divided into four groups: the control group, the aging group, the Rg1-treated aging group and the Rg1 anti-aging group. The expression of Sry genes in bone marrow cells of recipient mice was analyzed by fluorescence quantitative PCR, in order to determine the source of hematopoietic reconstruction cells, observe the survival time and the recovery of the hematology of peripheral blood, and study the reconstruction of the hematopoietic function of recipient mice, the hematopoietic recovery promoted by Rg1, the culture of CFU-Mix of hemopoietic progenitor cells, the cell cycle analysis and aging-related SA-beta-Gal staining analysis on biological characteristics of Sca-1 (+) HSC/HPC aging, and the effect of Rg1 in vivo regulation on Sca-1 + HSC/HPC aging. RESULT: The hematopoietic reconstruction cells of female recipient mice were derived from male donor mice. With the serial transplantation, the 30-day survival rate and the hematology in peripheral blood of recipient mice decreased. Sca-1 (+) HSC/HPC showed aging characteristics: the ratio of cells in G0/G1 phase and the positive rate of SA-beta-gal staining increased, whereas the number of CFU-Mix decreased. Compared with the aging group of the same generation, Rg1 -treated aging group and Rg1 anti-aging group showed higher 30-day survival rate and WBC, HCT, PLT and CFU-Mix, and lower cell ratio in Sca-1 (+) HSC/HPC G0/G1 stage and positive rate of SA-beta-gal staining. The Rg1 anti-aging group showed more significant changes than the Rg1 -treated aging group. CONCLUSION: Ginsenoside Rg1 has the effect of delaying and treating Sca-1 (+) HSC/HPC aging during the serial transplantation. Rg1 's anti-aging effect is superior to its effect of treating aging.

A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia.

Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.

Upregulation of nuclear factor-kappaB expression by SLURP-1 is mediated by alpha7-nicotinic acetylcholine receptor and involves both ionic events and activation of protein kinases.

SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1) is a novel auto/paracrine cholinergic peptide that can bind to α(7)-nicotinic acetylcholine receptor (nAChR), a high Ca(2+)-permeable ion channel coupled to regulation of nuclear factor-κB (NF-κB) expression. Elucidation of intracellular signaling events elicited by SLURP-1 is crucial for understanding the molecular mechanism of functioning of this novel hormone-like peptide that alters vital cell functions and can protect from tumorigenic transformation. In this study, we sought to dissect out the role of α(7)-nAChR in mediating the biologic effects of recombinant SLURP-1 on the immortalized line of human oral keratinocytes Het-1A. A multifold upregulation of the NF-κB expression at the mRNA and protein levels by SLURP-1 was only slightly diminished due to elimination of Na(+), whereas in Ca(2+)-free medium the effect of SLURP-1 was inhibited by >50%. Both in the absence of extracellular Ca(2+) and in the presence of Cd(2+) or Zn(2+), the SLURP-1-dependent elevation of NF-κB was almost completely blocked by inhibiting MEK1 activity. Downstream of α(7)-nAChR, the SLURP-1 signaling coupled to upregulation of NF-κB also involved Jak2 as well as Ca(2+)/calmodulin-dependent kinase II (CaMKII) and protein kinase C (PKC), whose inhibition significantly (P < 0.05) reduced the SLURP-1-induced upregulation of NF-κB. The obtained results indicated that activation of α(7)-nAChR by SLURP-1 leads to upregulation of the NF-κB gene expression due to activation of the Raf-1/MEK1/ERK1/2 cascade that proceeds via two complementary signaling pathways. One is mediated by the Ca(2+)-entry dependent CaMKII/PKC activation and another one by Ca(2+)-independent involvement of Jak2. Thus, there exists a previously not appreciated network of noncanonical auto/paracrine ligands of nAChR of the Ly-6 protein family, which merits further investigations.

Calreticulin maintains the low threshold of peptide required for efficient antigen presentation.

Calreticulin (CRT) plays a critical role in MHC class I antigen processing and elicits peptide-specific CD8(+) T cell responses against tumours when administered with peptides. However, how CRT contributes to class I antigen processing and the mechanism of its adjuvant effect in anti-tumour responses, remain to be elucidated. Here we show that reduced class I expression in CRT deficient cells can be restored by the direct delivery of peptides into the ER or by incubation at low temperature. CRT deficient cells exhibited a TAP-deficient phenotype in terms of class I assembly, without loss of TAP expression or functionality. Furthermore, a higher concentration of antigen in the cytosol is required for specific T cell stimulation, suggesting that CRT has a functional role in the maintenance of the low peptide concentration threshold required in the ER for efficient antigen presentation. In the absence of CRT, ERp57 is up-regulated, which indicates that they collaborate with each other in class I antigen processing.

Sca-1 and Thy-1 accelerate neuron-like differentiation in bone marrow stromal cells.

Bone marrow stromal cells taken from EGFP transgenic mice were sorted by magnetic beads with surface markers for Sca-1 and Thy-1. The cells were then co-cultured on organotypic hippocampal slice or with neuronal cell feeder in dish. On hippocampus, both Sca-1 and Thy-1 positive cells showed 4- 8 folds higher potential to show neuron-like morphology than negative cells. In dish, negative cells fewly survived but each positive cells survived and showed neuron-like differentiation. In both culture condition, retinoic acid supplement accelerate differentiation. Differentiated Sca-1 and Thy-1 positive cells were immunohistochemically GFAP- and NeuN-negative but nestin-, neurofilament- and NSE-positive. Neuron-like differentiation of bone marrow cells can be enhanced by selection using cell surface proteins.

Bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells do not contribute to vasculogenesis in Lewis lung carcinoma.

The development of tumor vasculature is thought to occur through two complementary processes: sprouting angiogenesis from preexisting blood vessels of the host, and vasculogenesis, which involves the spontaneous development of vessels through specific recruitment, differentiation, and vascular incorporation of circulating endothelial cells (EC), endothelial progenitor cells (EPC), or potentially bone marrow-derived cells. Recent reports, however, have challenged the belief that bone marrow-derived cells contribute to tumor neovascularization, claiming an exclusive role for sprouting angiogenesis in tumor blood vessel development. In the present study, we explored the recruitment behavior of bone marrow-derived lin(-)c-kit(+)Sca-1+ stem cells to subcutaneously implanted Lewis lung carcinoma in a syngeneic bone marrow transplantation model. We observed that although lin(-)c-kit(+)Sca-1+ and their derived cells demonstrate significant recruitment to carcinomas in vivo, they do not appear to functionally contribute to tumor neovascularization. Furthermore, our results support the hypothesis that new vessel formation in carcinomas occurs primarily through endothelialization from adjacent and preexisting vasculature.

Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.

The asymmetry in idiotype-isotype expression in the response to phosphocholine is due to divergence in the expressed repertoires of Lyb-5+ and Lyb-5- B cells.

The X-linked CBA/N immune defect was used to investigate the roles of Lyb-5- and Lyb-5+ B cells in the memory response to PC-KLH (phosphocholine-conjugated keyhole limpet hemocyanin). (CBA/N X BALB/c)F1 (CB) male mice express the xid mutation and thereby lack the Lyb-5+ B cell subset, whereas their female littermates are normal and express both Lyb-5+ and Lyb-5- B cells. After priming with PC-conjugated hemocyanin (PC-Hy) in complete Freund's adjuvant, female B cells produce three phenotypic sets of PC-KLH-specific antibody. The first set (group I) is dominated by T15+, IgM, IgA, and IgG3, PC-specific antibodies. The second subset (group II) is specific for phenylphosphocholine (PPC), and is dominated by T15-, IgG1, and IgG2 antibodies. The third set (group III) recognizes an epitope(s) composed of both the PPC hapten and carrier determinants. PC-Hy-primed B cells from immune defective CB male mice produce the same number of IgG1 and IgG2 plaque-forming cells (PFC) as do PC-Hy-primed normal female cells, and these PFC are also predominantly T15- and PPC specific (group II). In addition, a significant amount of group III IgG1 and IgG2 antibody is observed in the immune defective male response. In contrast to female B cells, immune defective male B cells produce a low IgM, IgA, and IgG3 memory response that is not composed of PC-specific (group I) antibodies; in fact, most of these antibodies arise from group III precursors and are not inhibited by either PC or PPC. PC-specific antibodies usually represent less than 25% of the anti-PC-KLH response in immune defective mice; however, these PC-specific antibodies are predominantly T15-. These data suggest that the Lyb-5-B cells in both normal and immune defective mice produce the T15-, IgG1, and IgG2 antibodies that dominate the secondary immune response to PC-KLH, and that the Lyb-5+ B cells produce the T15+, IgM, IgA, and IgG3 portion of the secondary response in normal mice. This hypothesis was confirmed by priming normal mice with the R36a strain of Streptococcus pneumoniae or with PC-Hy in saline. These forms of PC-antigen prime only the Lyb-5+ B cell subset. The adoptive transfer of these two B cell sources results in an anti-PC-KLH response that is T15 dominant and totally PC inhibitable.(ABSTRACT TRUNCATED AT 400 WORDS)