C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages.

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 -/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 -/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 -/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 -/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 -/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 -/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 -/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 -/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

Collectin 11 has a pivotal role in host defense against kidney and bladder infection in mice.

The urinary tract is constantly exposed to microorganisms. Host defense mechanisms in protection from microbial colonization and development of urinary tract infections require better understanding to control kidney infection. Here we report that the lectin collectin 11 (CL-11), particularly kidney produced, has a pivotal role in host defense against uropathogen infection. CL-11 was found in mouse urine under normal and pathological conditions. Mice with global gene ablation of Colec11 had increased susceptibility to and severity of kidney and to an extent, bladder infection. Mice with kidney-specific Colec11 ablation exhibited a similar disease phenotype to that observed in global Colec11 deficient mice, indicating the importance of kidney produced CL-11 for protection against kidney and bladder infection. Conversely, intravesical or systemic administration of recombinant CL-11 reduced susceptibility to and severity of kidney and bladder infection. Mechanism analysis revealed that CL-11 can mediate several key innate defense mechanisms (agglutination, anti- adhesion, opsonophagocytosis), and limit local inflammatory responses to pathogens. Furthermore, CL-11-mediated innate defense mechanisms can act on clinically relevant microorganisms including multiple antibiotic resistant strains. CL-11 was detectable in eight of 24 urine samples from patients with urinary tract infections but not detectable in urine samples from ten healthy individuals. Thus, our findings demonstrate that CL-11 is a key factor of host defense mechanisms in kidney and bladder infection with therapeutic potential for human application.

Collectin-11 promotes cancer cell proliferation and tumor growth.

Collectin-11 (CL-11) is a recently described soluble C-type lectin that has distinct roles in embryonic development, host defence, autoimmunity, and fibrosis. Here we report that CL-11 also plays an important role in cancer cell proliferation and tumor growth. Melanoma growth was found to be suppressed in Colec11-/- mice in a s.c. B16 melanoma model. Cellular and molecular analyses revealed that CL-11 is essential for melanoma cell proliferation, angiogenesis, establishment of more immunosuppressive tumor microenvironment, and the reprogramming of macrophages to M2 phenotype within melanomas. In vitro analysis revealed that CL-11 can activate tyrosine kinase receptors (EGFR, HER3) and ERK, JNK, and AKT signaling pathways and has a direct stimulatory effect on murine melanoma cell proliferation. Furthermore, blockade of CL-11 (treatment with L-fucose) inhibited melanoma growth in mice. Analysis of open data sets revealed that COLEC11 gene expression is upregulated in human melanomas and that high COLEC11 expression has a trend toward poor survival. CL-11 also had direct stimulatory effects on human tumor cell proliferation in melanoma and several other types of cancer cells in vitro. Overall, our findings provide the first evidence to our knowledge that CL-11 is a key tumor growth-promoting protein and a promising therapeutic target in tumor growth.

The Negative Impact of Triptolide on the Immune Function of Human Natural Killer Cells.

Triptolide (TP), a bioactive compound extracted the from traditional Chinese medicine Tripterygium wilfordii Hook F (TwHF), has been shown to be effective in treating several autoimmune diseases, and has suppressive effects in several key immune cells such as dendritic cells, T cells, and macrophages. However, it is unknown whether TP has an impact on natural killer (NK) cells. Here, we report that TP has suppressive effects on human NK cell activity and effector functions. The suppressive effects were observed in human peripheral blood mononuclear cell cultures and purified NK cells from healthy donors, as well as in purified NK cells from patients with rheumatoid arthritis. TP treatment induced downregulation of NK-activating receptor (CD54, CD69) expression and IFN-gamma secretion, in a dose-dependent manner. When exposed to K562 target cells, TP treatment induced inhibition of surface expression of CD107a and IFN-gamma synthesis in NK cells. Furthermore, TP treatment induced activation of inhibitory signaling (SHIP, JNK) and inhibition of MAPK signaling (p38). Thus, our findings demonstrate a previously unknown role for TP in NK cell functional suppression and reveal several key intracellular signaling that can be regulated by TP. Our findings also offer new insight into mechanisms of TP therapeutic treatment in autoimmune disease.

Protective role for C3aR in experimental chronic pyelonephritis.

Emerging evidence suggest that C3aR plays important roles in homeostasis, host defense and disease. Although it is known that C3aR is protective in several models of acute bacterial infections, the role for C3aR in chronic infection is largely unknown. Here we show that C3aR is protective in experimental chronic pyelonephritis. Global C3aR deficient (C3ar-/- ) mice had higher renal bacterial load, more pronounced renal histological lesions, increased renal apoptotic cell accumulation, tissue inflammation and extracellular matrix deposition following renal infection with uropathogenic E. coli (UPEC) strain IH11128, compared to WT control mice. Myeloid C3aR deficient (Lyz2-C3ar-/- ) mice exhibited a similar disease phenotype to global C3ar-/- mice. Pharmacological treatment with a C3aR agonist reduced disease severity in experimental chronic pyelonephritis. Furthermore, macrophages of C3ar-/- mice exhibited impaired ability to phagocytose UPEC. Our data clearly demonstrate a protective role for C3aR against experimental chronic pyelonephritis, macrophage C3aR plays a major role in the protection, and C3aR is necessary for phagocytosis of UPEC by macrophages. Our observation that C3aR agonist curtailed the pathology suggests a therapeutic potential for activation of C3aR in chronic infection.

Human CD3+CD56+NKT-like cells express a range of complement receptors and C3 activation has negative effects on these cell activity and effector function.

CD3+CD56+NKT-like cells are a rare population of lymphocytes that serve important roles in various types of immune-related diseases, and particularly in cancer. The complement system regulates inflammatory and immune responses by interacting with complement receptors expressed on a range of immune cells. However, whether CD3+CD56+NKT-like cells are regulated by the complement system has still not been definitively determined. In the present study, the expression of complement receptors and regulators in gated CD3+CD56+NKT-like cells isolated from human peripheral blood was assessed using PCR and flow cytometry. The results showed that human CD3+CD56+NKT-like cells expressed a range of complement receptors and regulators, such as CR3, C3aR, C5aR, C5L2, CD46 and CD55. Furthermore, the presence of complement component 3 (C3), a key component in complement activation in culture supernatant, mitigated the activity, IFN-γ production and killing function of CD3+CD56+NKT-like cells. The present study provides evidences supporting the relationship between complement activation and functional modulation of CD3+CD56+NKT-like cells, expanding our knowledge of the complement regulatory network, and also highlighting a potential target for treatment of numerous immune-related diseases, particularly NKT cell-based tumor adoptive immunotherapy.

Fibroblast growth factor inducible 14 signaling facilitates anti-dsDNA IgG penetration into mesangial cells.

Anti-double-stranded DNA (dsDNA) antibodies induce renal damage in patients with systemic lupus erythematosus by triggering fibrotic processes in kidney cells. However, the precise mechanism underlying penetration of anti-dsDNA immunoglubolin G (IgG) into cells remains unclear. This study was designed to investigate the effect of tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor inducible 14 (Fn14) signaling on anti-dsDNA IgG penetration into cells. Mesangial cells were cultured in vitro, and stimulated with TWEAK and anti-dsDNA IgG. The results revealed that TWEAK dose-dependently enhanced cellular internalization of anti-dsDNA IgG and the expression of high-mobility group box 1 (HMGB1). In addition, TWEAK and anti-dsDNA IgG synthetically downregulate suppressor of cytokine signaling 1, and induce the expression of various fibrotic factors. Furthermore, inhibition of HMGB1 attenuates the enhancement effect of TWEAK on anti-dsDNA IgG internalization. The TWEAK upregulation of HMGB1 involves the nuclear factor-κB and phosphatidylinositide 3-kinase/protein kinase B pathways. Therefore, TWEAK/Fn14 signaling contributes to the penetration of anti-dsDNA IgG and relevant fibrotic processes in mesangial cells.

Inhibition of suppressor of cytokine signaling 1 mediates the profibrotic effect of TWEAK/Fn14 signaling on kidney cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) engagement with the receptor Fn14 contributes to the fibrotic process of kidney cells in systemic lupus erythematosus. Downregulation of the protein suppressor of cytokine signaling 1 (SOCS1) correlates with amplified production of proinflammatory factors and cell apoptosis, which participate in the pathogenesis of lupus nephritis. To elucidate the potential role of SOCS1 in TWEAK/Fn14 signaling, we determined the SOCS1 levels in primary kidney cells from MRL/MpJ (control strain) or MRL/lpr (lupus-prone) mice. These cells (mesangial cells, glomerular endothelial cells, and tubular epithelial cells) were also evaluated after stimulation with TWEAK (0 to 250 ng/mL). The results showed that the lupus-prone cells exhibited reduced SOCS1 expression. TWEAK induced the production of profibrotic factors (laminin, fibronectin, (CC motif) ligand 20, etc.) in kidney cells from both mouse strains. TWEAK stimulation also decreased both the mRNA and protein levels of SOCS1 in all cells. Moreover, the effect of TWEAK on mesangial cells was amplified by pre-transfection of SOCS1 siRNA but was partly reduced with SOCS1 overexpression by adenoviral delivery. Therefore, TWEAK/Fn14 activation contributes to renal fibrosis in lupus nephritis involving the depression of SOCS1 function.

Fn14 Deficiency Ameliorates Anti-dsDNA IgG-Induced Glomerular Damage in SCID Mice.

Many studies have demonstrated that anti-dsDNA IgG is closely associated with lupus nephritis. Recently, it was found that activation of the fibroblast growth factor-inducible 14 (Fn14) signaling pathway damages glomerular filtration barrier in MRL/lpr lupus-prone mice. However, MRL/lpr mice have high titers of serum autoantibodies other than anti-dsDNA IgG. The aim of this study was to further explore the effect of Fn14 deficiency on anti-dsDNA IgG-induced glomerular damage in severe combined immunodeficiency (SCID) mice that have no endogenous IgG. Fn14 deficiency was generated in SCID mice. The murine hybridoma cells producing control IgG or anti-dsDNA IgG were intraperitoneally injected into mice. In two weeks, the urine, serum, and kidney tissue samples were harvested from mice at sacrifice. It showed that the injection of anti-dsDNA IgG, but not control IgG hybridoma cells, induced proteinuria and glomerular damage in SCID mice. Between the wild-type (WT) and knockout (KO) mice injected with anti-dsDNA IgG hybridoma cells, the latter showed a decrease in both proteinuria and glomerular IgG deposition. The histopathological changes, inflammatory cell infiltration, and proinflammatory cytokine production were also attenuated in the kidneys of the Fn14-KO mice upon anti-dsDNA IgG injection. Therefore, Fn14 deficiency effectively protects SCID mice from anti-dsDNA IgG-induced glomerular damage.

Complement Receptor 3 Has Negative Impact on Tumor Surveillance through Suppression of Natural Killer Cell Function.

Complement receptor 3 (CR3) is expressed abundantly on natural killer (NK) cells; however, whether it plays roles in NK cell-dependent tumor surveillance is largely unknown. Here, we show that CR3 is an important negative regulator of NK cell function, which has negative impact on tumor surveillance. Mice deficient in CR3 (CD11b-/- mice) exhibited a more activated NK phenotype and had enhanced NK-dependent tumor killing. In a B16-luc melanoma-induced lung tumor growth and metastasis model, mice deficient in CR3 had reduced tumor growth and metastases, compared with WT mice. In addition, adaptive transfer of NK cells lacking CR3 (into NK-deficient mice) mediated more efficient suppression of tumor growth and metastases, compared with the transfer of CR3 sufficient NK cells, suggesting that CR3 can impair tumor surveillance through suppression of NK cell function. In vitro analyses showed that engagement of CR3 with iC3b (classical CR3 ligand) on NK cells negatively regulated NK cell activity and effector functions (i.e. direct tumor cell killing, antibody-dependent NK-mediated tumor killing). Cell signaling analyses showed that iC3b stimulation caused activation of Src homology 2 domain-containing inositol-5-phosphatase-1 (SHIP-1) and JNK, and suppression of ERK in NK cells, supporting that iC3b mediates negative regulation of NK cell function through its effects on SHIP-1, JNK, and ERK signal transduction pathways. Thus, our findings demonstrate a previously unknown role for CR3 in dysregulation of NK-dependent tumor surveillance and suggest that the iC3b/CR3 signaling is a critical negative regulator of NK cell function and may represent a new target for preserving NK cell function in cancer patients and improving NK cell-based therapy.

TWEAK/Fn14 Activation Participates in Ro52-Mediated Photosensitization in Cutaneous Lupus Erythematosus.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) binds to its sole receptor fibroblast growth factor-inducible 14 (Fn14), participating in various inflammatory responses. Recently, TWEAK/Fn14 activation was found prominent in the lesions of cutaneous lupus erythematosus (CLE). This study was designed to further reveal the potential role of this pathway in Ro52-mediated photosensitization. TWEAK, Fn14, and Ro52 were determined in the skin lesions of patients with CLE. Murine keratinocytes received ultraviolet B (UVB) irradiation or plus TWEAK stimulation and underwent detection for Ro52 and proinflammatory cytokines. The chemotaxis of J774.2 macrophages was evaluated on TWEAK stimulation of cocultured keratinocytes. We found that TWEAK, Fn14, and downstream cytokines were highly expressed in CLE lesions that overexpressed Ro52. Moreover, TWEAK enhanced the UVB-induced Ro52 upregulation in murine keratinocytes. Meanwhile, TWEAK stimulation of keratinocytes favored the migration of macrophages through promoting the production of chemokine C-C motif ligands 17 and 22. Furthermore, Fn14 siRNA transfection or nuclear factor-kappa B (NF-κB) inhibitor abrogated the TWEAK enhancement of Ro52 expression in keratinocytes. Similarly, TNF receptor associated factor 2 (TRAF2) siRNA reduced the protein level of Ro52 in these cells upon TWEAK stimulation. Interestingly, UVB irradiation increased the expression of TNF receptor type 1 (TNFR1) but not affecting TNFR2 expression in keratinocytes. In conclusion, the TWEAK/Fn14 signaling participates in Ro52-mediated photosensitization and involves the activation of NF-κB pathway as well as the function of the TRAF2/TNFR partners.

IL-25 attenuates rheumatoid arthritis through suppression of Th17 immune responses in an IL-13-dependent manner.

IL-25, a new member of the IL-17 cytokine family, is involved in type 2 immunity initiation and has been associated with the pathogenesis of rheumatoid arthritis (RA). However, its exact role remains unclear. Here, we aimed to analyse IL-25 expression in the serum and synovial fluid of RA patients and evaluated the correlations between serum IL-25 levels, clinical and laboratory values and inflammation cytokines. Additionally, we investigated whether IL-25 can suppress Th1/Th17 responses involved in RA pathogenesis. We further determined whether IL-25 can alleviate collagen-induced arthritis (CIA) development in mice and the underlying mechanisms using in vitro and in vivo experiments. Our results showed that IL-25 was upregulated in the serum and synovial fluid of RA patients. Increased serum IL-25 levels were associated with disease severity and inflammatory response in RA patients. Furthermore, IL-25 inhibited CD4+ T-cell activation and differentiation into Th17 cells, without affecting Th1 cells in human RA and CIA models. Administration of IL-25 could attenuate CIA development by Th17 suppression in an IL-13-dependent manner. Our findings indicate that IL-25 plays a potent immunosuppressive role in the pathogenesis of RA and CIA by downregulating Th17 cell response, and thus, may be a potential therapeutic agent for RA.

Expression and regulation of complement receptors by human natural killer cells.

Integration of cellular and humoral arms of the innate immune response is fundamental to the development of powerful effector functions in host defence as well as aberrant immune responses. Here, we provide evidence in support of the relationship between complement activation and NK cell functional modulation. We demonstrate that human NK cells and both CD56(bright)CD16(-) and CD56(dim)CD16(+) populations express receptors known to detect the biologically active peptides C3a and C5a (i.e. C3aR, C5aR, C5L2) and the covalently-bound fragments C3b and metabolites iC3b and C3d which serve in immune adhesion (e.g. CR3, CR4). We also show that several pathogen- or tumour/inflammation-related stimuli differentially regulated those complement receptor expression. Furthermore, our results suggest that C3 fragments (C3a, iC3b) have a negative regulatory effect on IFN-γ production in NK cells. This work provides extensive information of human complement receptors relevant to the integrated actions of complement and NK cells which has been suggested by animal studies. The observations may act as a resource that allows further understanding and exploitation of role of complement in human health and immune mediated diseases.

The prognostic value of CXCR4 in ovarian cancer: a meta-analysis.

OBJECTIVE: Recent reports have shown that C-X-C chemokine receptor 4 (CXCR4) is expressed in ovarian cancer and plays an important role in metastasis. However, the prognostic value of CXCR4 in ovarian cancer remains controversial and has not been emphasized. The aim of this study is to evaluate the prognostic significance of CXCR4 in ovarian cancer by performing a meta-analysis. METHODS: We systematically searched for studies evaluating the relationship between CXCR4 expression and the outcome of ovarian cancer patients. Only articles in which CXCR4 expression was detected by immunohistochemical staining were included. Hazard ratios (HRs) and relative risk (RR) with 95% confidence intervals (CIs) were pooled as effect size (ES) across studies for overall survival (OS) and progression-free survival (PFS). RESULTS: A total of 729 patients from 7 studies (6 articles) were included in this meta-analysis. Our results showed that high CXCR4 expression was significantly associated with poor prognosis in terms of OS (ES, 2.81; 95% CI, 1.16-6.80; p = 0.022) and PFS (ES, 8.48; 95% CI, 2.13-33.70; p = 0.002) in ovarian cancer patients. The association between high CXCR4 expression and poor ovarian cancer prognosis in OS was also statistically significant in subgroups of Asian and III-IV patients constituting 70%. CONCLUSIONS: The present meta-analysis indicated that high CXCR4 expression was associated with poor prognosis in ovarian cancer. More studies, especially larger scale and well-matched researches, are warranted to clarify the prognostic effect of CXCR4 on the outcome of ovarian cancer.

Prognostic value of cancer stem cell marker aldehyde dehydrogenase in ovarian cancer: a meta-analysis.

OBJECTIVE: Aldehyde dehydrogenase (ALDH) has recently been reported as a marker of cancer stem-like cells in ovarian cancer. However, the prognostic role of ALDH in ovarian cancer still remains controversial. In this study, we aimed to evaluate the association between the expression of ALDH and the outcome of ovarian cancer patients by performing a meta-analysis. METHODS: We systematically searched for studies investigating the relationships between ALDH expression and outcome of ovarian cancer patients. Only articles in which ALDH expression was detected by immunohistochemical staining were included. A meta-analysis was performed to generate combined hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS) and disease-free survival (DFS). RESULTS: A total of 1,258 patients from 7 studies (6 articles) were included in the analysis. Our results showed that high ALDH expression in patients with ovarian cancer was associated with poor prognosis in terms of Os (HR, 1.25; 95% CI, 1.07-1.47; P = 0.005) and DFS (HR, 1.58; 95% CI, 1.00-2.49; P = 0.052), though the difference for DFS was not statistically significant. In addition, there was no evidence of publication bias as suggested by Begg's and Egger's tests (Begg's test, P = 0.707; Egger's test, P = 0.355). CONCLUSION: The present meta-analysis indicated that elevated ALDH expression was associated with poor prognosis in patients with ovarian cancer.