Staphylococcal Panton–Valentine Leucocidin and Gamma Haemolysin Target and Lyse Mature Bone Marrow Leucocytes

Staphylococcus aureus is a major human pathogen, inducing several infections ranging from the benign to the life-threatening, such as necrotising pneumonia. S. aureus is capable of producing a great variety of virulence factors, such as bicomponent pore-forming leucocidin, which take part in the physiopathology of staphylococcal infection. In necrotising pneumonia, Panton–Valentine leucocidin (PVL) induces not only lung injury and necrosis, but also leukopenia, regarded as a major factor of a poor prognosis. The aim of the present study was to evaluate the effect of bicomponent pore-forming leucocidin, PVL and gamma haemolysin on bone marrow leucocytes, to better understand the origin of leukopenia. Using multi-parameter cytometry, the expression of leucocidin receptors (C5aR, CXCR1, CXCR2, and CCR2) was assessed and toxin-induced lysis was measured for each bone marrow leucocyte population. We observed that PVL resulted in myeloid-derived cells lysis according to their maturation and their C5aR expression; it also induced monocytes lysis according to host susceptibility. Haemolysin gamma A, B, and C (HlgABC) displayed cytotoxicity to monocytes and natural killer cells, hypothetically through CXCR2 and CXCR1 receptors, respectively. Taken together, the data suggest that PVL and HlgABC can lyse bone marrow leucocytes. Nevertheless, the origin of leukopenia in severe staphylococcal infection is predominantly peripheral, since immature cells stay insensitive to leucocidins.

C5aR correlated with the dissemination capacity of circulating tumor cells in hepatocellular carcinoma by targeting INHBA-p-smad2/3-EMT/MMPs axis.

e16649 Background: Circulating tumor cells (CTCs) play important role in tumor dissemination and is an independent survival predictor in Hepatocellular carcinoma (HCC) patients. The aim of this study was to investigate C5a/C5aR, an important axis in complement pathway, which causes the difference capacity of the dissemination of CTCs between HCC patients. The influence of C5a/C5aR axis on recurrence and HCC cell functions was also explored. Methods: Expression microarray analysis was carried out to identify key molecule that cause the difference of CTC enumeration. Clinical significance of the key gene C5aR was evaluated by immunohistochemistry in 187 resectable HCC patients with CTC detection from March 2011 to October 2014. The function of C5a/C5aR axis to enhance the dissemination capacity of CTCs was confirmed first in HCC cell line and than validated in a mouse model. CTCs isolated from the animal circulation were identified by immunostaining for human EpCAM and nuclear counterstaining of red blood cell-lysed blood. Results: Analysis of 187 HCC patients undergoing curative resection showed that C5aR high expression patients were prone to developing vascular invasion, suffering higher AFP level as well as higher percentage of recurrence and death. Further, C5aR expression was also positive correlated with CTC number in HCC patients. In vitro, we observed that cell migration, invasive, proliferation, anti-apoptosis ability and EMT could be greatly inhibited after C5aR knockdown. Opposite results could be observed when C5aR is overexpressed. Further exploration indicated that C5a/C5aR axis could upregulate the expression of INHBA/Activin, and induce phosphorylation of smad2/3 which maintains the mesenchymal phenotype in HCC. Inhibition of the C5a/C5aR signal pathway could decrease circulating capacities of tumor cells and inhibit their colonization to distal organs such as lung in mouse model. Conclusions: Our research elucidated a new C5a/C5aR targeted INHBA-p-smad2/3-EMT/MMP axis to regulate the dissemination of CTC in HCC patients. And these molecules could be novel therapeutic targets for inhibiting the metastasis of tumor cell in the futures.

N-Acetylcysteine Attenuates Cisplatin-Induced Acute Kidney Injury by Inhibiting the C5a Receptor

N-acetylcysteine has been widely used as a nutritional supplement and drug in humans for its antioxidant properties. The complement activation fragment C5a is a strong proinflammatory molecule that mediates cell adhesion, chemotaxis, and the complex biological functions. However, the effect of NAC on the C5a, and the relationship of those two with cisplatin-induced acute kidney injury are unknown. In cisplatin induced AKI mouse model, mice with NAC administration had a marked improvement in renal function (BUN and Cr), decreased pathological damage, reduced inflammation, and alleviated renal oxidative stress. Furthermore, C5a and C5aR expression in the cisplatin-treated group was notably increased compared with the control group, and this increase could be significantly inhibited by NAC. In addition, neutrophils coexpressed distinctly with C5aR, and the number of infiltrating neutrophils (MPO+ly6G+) and inflammatory factors decreased with NAC treatment in the cisplatin-treated group. Overall, these data demonstrate that NAC could ameliorate cisplatin-induced nephrotoxicity in mice and the protective effects may be conducted by inhibiting the activation of kidney inflammation and the complement system.

Apigenin inhibits C5a-induced proliferation of human nasopharyngeal carcinoma cells through down-regulation of C5aR

Complement 5a (C5a) is able to induce the proliferation of human nasopharyngeal carcinoma (NPC) cells. Therefore, an effective method or drug that can specifically inhibit C5a-induced proliferation of human NPC cells needs to be developed. Reportedly, Apigenin has antiproliferative effects on a variety of cancer cells. However, the effect of Apigenin on NPC cell proliferation and its underlying mechanism are still unclear. Herein, the present study aimed to evaluate the effect of Apigenin on C5a-induced proliferation of human NPC cells and its possible mechanism through down-regulation of C5aR. We revealed that Apigenin in vitro could not only inhibit proliferation of NPC cells and but also reduce the expression of C5aR and P300/CBP-associated factor (PCAF) as well as the activation of signal transducer and activator of transcription 3 (STAT3) in NPC cells. Furthermore, Apigenin reduced the proliferation of human NPC cells triggered by C5a through negative regulation of C5aR/PCAF/STAT3 axis. These might provide a new insight into the function of Apigenin in cancer treatment, and also provide a potential strategy for treating human NPC through inhibition of C5aR expression on cancer cells.

A Novel Role of Complement Components C1q and C5a in Hematopoietic Stem/Progenitor Cell Migration.

The complement protein C1q primarily provides a recognition and activation signal that triggers the classical pathway of complement, but it also has multiple immune functions including acting as a chemoattractant for neutrophils, eosinophils, mast and monocyte-derived dendritic cells. Moreover, hematopoietic stem cells have recently been shown to express the C1q receptor, C1qRp. C5a also strongly chemoattracts monocyte/macrophages to inflammatory sites. Recently we showed that G-CSF mobilization activates complement by a classical IgM-dependent pathway (Blood2005;106:1976a). In this study we examined the possible roles of C1q and C5a in hematopoietic stem/progenitor cells (HSPC) migration. We found that C5aR was expressed at the mRNA level on mobilized peripheral blood (mPB) and cord blood (CB) CD34+ cells but at the protein level only on mPB CD34+ cells. Flow cytometry revealed high C1qRp expression on both mPB and CB CD34+ cells. When the expression of C5aR and C1qRp was examined on the CB CD34+ cells expanded towards myeloid, megakaryocytic and erythroid lineages (at days 0, 3, 6, 11 and 14 of expansion), we found that C5aR expression increased with cell maturation (days 3–14) in both myeloid and megakaryocytic progenitors. In contrast, C1qRp was highly expressed on day 0, stayed constant in myeloid and megakaryocytic cells (days 3–11), and was down-regulated by day 14 in megakaryocytic cells. C1qRp was down-regulated in erythroid precursors during their maturation. We also found that C5a but not C1q is a chemoattractant for mPB CD34+ cells. In chemotaxis assays towards an SDF-1 gradient, C5a primed chemotactic responses of both mPB and CB CD34+ cells to a low (10 ng/mL) gradient of SDF-1 (up to 80% of their response to a high (200 ng/mL) SDF-1 gradient); C1q primed the chemotactic responses of both types of CD34+ cells (up to 100% of the response to a high SDF-1 gradient), and the priming effect of C1q on SDF-1-induced chemotaxis of expanded myeloid and megakaryocytic precursors decreased, consistent with down-regulation of C1qRp on these cells by day 14. Hence these results indicate that HSPC and progenitor cells express functional C5a and C1q receptors and that both the C5a-C5aR and C1q-C1qRp axes sensitize the responses of these cells to SDF-1 and thus could play a role in HSPC homing/mobilization to bone marrow. Further studies in animal models are needed to elucidate the roles of C5a and C1q in HSPC trafficking.