Immortalized Human Conjunctival Epithelial Cells Produce Functional Complement C3 and C4 Proteins.

PURPOSE: The aim of this study was to assess whether complement proteins C3 and C4 are produced by immortalized human conjunctival epithelial (HCjE) cells. METHODS: Supernatants and cell lysates from undifferentiated and differentiated HCjE cells were assayed for C3 and C4 by enzyme-linked immunosorbent assay. To measure complement protein function, supernatants and lysates were treated with heat-aggregated IgG, and soluble C5b-9 was measured. RESULTS: C3 was upregulated in supernatants from differentiated HCjE cells compared with undifferentiated HCjE cells (556.55 ± 91.75 vs. 56.95 ± 12.09 ng/mL, P <0.001). C4 was also increased in supernatants but to a much lesser extent (0.599 ± 0.476 vs. 0.172 ± 0.0133 ng/mL, P = 0.03). From HCjE cell lysates, total C3 production was 9.03 times higher in differentiated HCjE cells ( P <0.001), whereas total C4 remained relatively unchanged. After activation with heat-aggregated IgG, sC5b-9 could be detected from both undifferentiated and differentiated HCjE cell lysates, but not in the HCjE supernatants. CONCLUSIONS: HCjE cells produce C3 and C4 in sufficient quantities to support the formation of sC5b-9, confirming their biological activity and suggesting that HCjE cells likely produce all complement proteins C1 through C9.

Clustering Patients With Gout Based on Comorbidities and Biomarkers: A Cross-Sectional Study.

OBJECTIVE: This single-center clinical study identifies clusters of different phenotypes and pathophysiology subtypes of patients with gout and associated comorbidities. METHODS: Patients clinically diagnosed with gout were enrolled between January 2018 and December 2019. Hierarchical cluster analyses were performed using clinical data or biological markers, inflammatory markers, and oxidative stress pathway metabolites assayed from serum and plasma samples. Subgroup clusters were compared using ANOVA for continuous data and chi-square tests for categorical data. RESULTS: Hierarchical cluster analysis identified 3 clusters. Cluster 1 (C1; n = 24) comprised dyslipidemia, hypertension, and early-onset gout, without tophi. Cluster 2 (C2; n = 25) comprised hypertension, dyslipidemia, nephrolithiasis, and obesity. Cluster 3 (C3; n = 39) comprised multiple comorbidities and tophi. Post hoc comparisons of data obtained from samples of patients in C1, C2, and C3 revealed significant differences in the levels of oxidative stress and inflammation-related markers, including 3-nitrotyrosine, tumor necrosis factor, C-reactive protein, interleukin (IL) 1ß, IL-6, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Reclustering patients based on all markers as well as on the biological markers that significantly differed among the initial clusters identified similar clusters. CONCLUSION: Oxidative stress and inflammatory marker levels may affect the development and clinical manifestations (ie, clinical phenotypes) of gout. Measuring oxidative stress and levels of inflammatory cytokines is a potential adjunctive tool and biomarker for early identification and management of gout.

Decreased Levels of STAT1 and Interferon-γ-Induced STAT1 Phosphorylation in Rheumatoid Arthritis CD4 and CD8 T Cells.

OBJECTIVE: We investigated whether a previously reported association of IFNGR expression with rheumatoid arthritis (RA) and its radiographic severity reflects differences in proximal interferon-γ (IFN-γ) signaling in T cells from patients with RA compared with healthy controls (HC). METHODS: Using phosphoflow cytometry, we compared IFN-γ-stimulated signal transducer and activator of transcription 1 (STAT1) activation in CD4+ and CD8+ T-cell populations from patients with RA and HC. RESULTS: Compared with controls, patients with RA had a higher proportion of CD4+ T cells, associated with expansion of the CD4+ effector memory subset. Several CD4+ T-cell types exhibited reduced IFN-γ-induced phosphoSTAT1Y701 (pSTAT1Y701 ) in patients with RA compared with HC. Engaging the T-cell receptor (TCR) complex on CD4+ T cells during IFN-γ stimulation abrogated the reduction in STAT1 activation in patients with RA but had no effect in HC. The phosphorylation of STAT1S727 was similar in CD4+ T cells from patients with RA and HC. In contrast to CD4+ T cells, IFN-γ-induced pSTAT1Y701 levels in CD8+ T cells were equivalent or higher in patients with RA compared with HC. Total STAT1 levels (phosphorylated + unphosphorylated) were lower in CD4+ and CD8+ T cells from patients with RA compared with HC. CONCLUSION: We report diminished IFN-γ-induced pSTAT1Y701 levels in CD4+ T cells in patients with RA, which were restored by TCR engagement. There were lower levels of total STAT1 in patients with RA compared with HC, but this likely does not explain diminished IFN-γ-induced pSTAT1Y701 levels in CD4+ T cells because activation in CD8+ T cells was higher or equivalent to that seen in HC. The enhanced IFNGR expression in patients with RA reported previously may reflect a compensatory mechanism to overcome deficiency in IFN-γ responsiveness.

Therapeutic Lowering of C-Reactive Protein.

In the blood of healthy individuals C-reactive protein (CRP) is typically quite scarce, whereas its blood concentration can rise robustly and rapidly in response to tissue damage and inflammation associated with trauma and infectious and non-infectious diseases. Consequently, CRP plasma or serum levels are routinely monitored in inpatients to gauge the severity of their initial illness and injury and their subsequent response to therapy and return to health. Its clinical utility as a faithful barometer of inflammation notwithstanding, it is often wrongly concluded that the biological actions of CRP (whatever they may be) are manifested only when blood CRP is elevated. In fact over the last decades, studies done in humans and animals (e.g. human CRP transgenic and CRP knockout mice) have shown that CRP is an important mediator of biological activities even in the absence of significant blood elevation, i.e. even at baseline levels. In this review we briefly recap the history of CRP, including a description of its discovery, early clinical use, and biosynthesis at baseline and during the acute phase response. Next we overview evidence that we and others have generated using animal models of arthritis, neointimal hyperplasia, and acute kidney injury that baseline CRP exerts important biological effects. In closing we discuss the possibility that therapeutic lowering of baseline CRP might be a useful way to treat certain diseases, including cancer.

Mice expressing the variant rs1143679 allele of ITGAM (CD11b) show impaired DC-mediated T cell proliferation.

Genome-wide association studies (GWAS) and functional genomic analyses have implicated several ITGAM (CD11b) single-nucleotide polymorphisms (SNPs) in the development of SLE and other disorders. ITGAM encodes the αM chain of the ß2 integrin Mac-1, a receptor that plays important roles in myeloid cell functions. The ITGAM SNP rs1143679, which results in an arginine to histidine change at amino acid position 77 of the CD11b protein, has been shown to reduce binding to several ligands and to alter Mac-1-mediated cellular response in vitro. Importantly, however, the potential contribution of this SNP variant to the initiation and/or progression of immune and inflammatory processes in vivo remains unexplored. Herein, we describe for the first time the generation and characterization of a mouse line expressing the 77His variant of CD11b. Surprisingly, we found that 77His did not significantly affect Mac-1-mediated leukocyte migration and activation as assessed using thioglycollate-induced peritonitis and LPS/TNF-α-induced dermal inflammation models. In contrast, expression of this variant did alter T cell immunity, as evidenced by significantly reduced proliferation of ovalbumin (OVA)-specific transgenic T cells in 77His mice immunized with OVA. Reduced antigen-specific T cell proliferation was also observed when either 77His splenic dendritic cells (DCs) or bone marrow-derived DCs were used as antigen-presenting cells (APCs). Although more work is necessary to determine how this alteration might influence the development of SLE or other diseases, these in vivo findings suggest that the 77His variant of CD11b can compromise the ability of DCs to induce antigen-driven T cell proliferation.

C-Reactive Protein Promotes the Expansion of Myeloid Derived Cells With Suppressor Functions.

Previously we established that human C-reactive protein (CRP) exacerbates mouse acute kidney injury and that the effect was associated with heightened renal accumulation of myeloid derived cells with suppressor functions (MDSC). Herein we provide direct evidence that CRP modulates the development and suppressive actions of MDSCs in vitro. We demonstrate that CRP dose-dependently increases the generation of MDSC from wild type mouse bone marrow progenitors and enhances MDSC production of intracellular reactive oxygen species (iROS). When added to co-cultures, CRP significantly enhanced the ability of MDSCs to suppress CD3/CD28-stimulated T cell proliferation. Experiments using MDSCs from FcγRIIB deficient mice (FcγRIIB-/-) showed that CRP's ability to expand MDSCs and trigger their increased production of iROS was FcγRIIB-independent, whereas its ability to enhance the MDSC T cell suppressive action was FcγRIIB-dependent. Importantly, CRP also enabled freshly isolated primary human neutrophils to suppress proliferation of autologous T cells. These findings suggest that CRP might be an endogenous regulator of MDSC numbers and actions in vivo.

C-Reactive Protein Impairs Dendritic Cell Development, Maturation, and Function: Implications for Peripheral Tolerance.

C-reactive protein (CRP) is the prototypical acute phase reactant, increasing in blood concentration rapidly and several-fold in response to inflammation. Recent evidence indicates that CRP has an important physiological role even at low, baseline levels, or in the absence of overt inflammation. For example, we have shown that human CRP inhibits the progression of experimental autoimmune encephalomyelitis (EAE) in CRP transgenic mice by shifting CD4+ T cells away from the TH1 and toward the TH2 subset. Notably, this action required the inhibitory Fcγ receptor IIB (FcγRIIB), but did not require high levels of human CRP. Herein, we sought to determine if CRP's influence in EAE might be explained by CRP acting on dendritic cells (DC; antigen presenting cells known to express FcγRIIB). We found that CRP (50 µg/ml) reduced the yield of CD11c+ bone marrow-derived DCs (BMDCs) and CRP (≥5 µg/ml) prevented their full expression of major histocompatibility complex class II and the co-stimulatory molecules CD86 and CD40. CRP also decreased the ability of BMDCs to stimulate antigen-driven proliferation of T cells in vitro. Importantly, if the BMDCs were genetically deficient in mouse FcγRIIB then (i) the ability of CRP to alter BMDC surface phenotype and impair T cell proliferation was ablated and (ii) CD11c-driven expression of a human FCGR2B transgene rescued the CRP effect. Lastly, the protective influence of CRP in EAE was fully restored in mice with CD11c-driven human FcγRIIB expression. These findings add to the growing evidence that CRP has important biological effects even in the absence of an acute phase response, i.e., CRP acts as a tonic suppressor of the adaptive immune system. The ability of CRP to suppress development, maturation, and function of DCs implicates CRP in the maintenance of peripheral T cell tolerance.

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance.

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor FcγRIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking FcγRIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal. IgG transfer in IgG-deficient mice implicated IgG as the pathogenetic ligand for endothelial FcγRIIB in obesity-induced insulin resistance. Moreover, IgG transferred from patients with T2DM but not from metabolically healthy subjects caused insulin resistance in IgG-deficient mice via FcγRIIB, indicating that similar processes may be operative in T2DM in humans. Mechanistically, the activation of FcγRIIB by IgG from obese mice impaired endothelial cell insulin transcytosis in culture and in vivo. These effects were attributed to hyposialylation of the Fc glycan, and IgG from T2DM patients was also hyposialylated. In HFD-fed mice, supplementation with the sialic acid precursor N-acetyl-D-mannosamine restored IgG sialylation and preserved insulin sensitivity without affecting weight gain. Thus, IgG sialylation and endothelial FcγRIIB may represent promising therapeutic targets to sever the link between obesity and T2DM.

C-reactive protein promotes acute kidney injury via Smad3-dependent inhibition of CDK2/cyclin E.

Acute kidney injury (AKI) is exacerbated in C-reactive protein transgenic mice but alleviated in Smad3 knockout mice. Here we used C-reactive protein transgenic/Smad3 wild-type and C-reactive protein transgenic/Smad3 knockout mice to investigate the signaling mechanisms by which C-reactive protein promotes AKI. Serum creatinine was elevated, and the extent of tubular epithelial cell necrosis following ischemia/reperfusion-induced AKI was greater in C-reactive protein transgenics but was blunted when Smad3 was deleted. Exacerbation of AKI in C-reactive protein transgenics was associated with increased TGF-ß/Smad3 signaling and expression of the cyclin kinase inhibitor p27, but decreased phosphorylated CDK2 and expression of cyclin E. Concomitantly, tubular epithelial cell proliferation was arrested at the G1 phase in C-reactive protein transgenics with fewer cells entering the S-phase cell cycle as evidenced by fewer bromodeoxyuridine-positive cells. In contrast, the protection from AKI in C-reactive protein transgenic/Smad3 knockout mice was associated with decreased expression of p27 and promotion of CDK2/cyclin E-dependent G1/S transition of tubular epithelial cells. In vitro studies using tubular epithelial cells showed that C-reactive protein activates Smad3 via both TGF-ß-dependent and ERK/MAPK cross talk mechanisms, Smad3 bound directly to p27, and blockade of Smad3 or the Fc receptor CD32 prevented C-reactive protein-induced p27-dependent G1 cell cycle arrest. In vivo, treatment of C-reactive protein transgenics with a Smad3 inhibitor largely improved AKI outcomes. Thus, C-reactive protein may promote AKI by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27-driven inhibition of the CDK2/cyclin E complex. Targeting Smad3 may offer a new treatment approach for AKI.

C-reactive protein exacerbates renal ischemia-reperfusion injury: are myeloid-derived suppressor cells to blame?

Myeloid-derived suppressor cells (MDSCs) are a CD11b(+)Gr1(+) population in mice that can be separated into granulocytic (g-MDSC) and monocytic (m-MDSC) subtypes based on their expression of Ly6G and Ly6C. Both MDSC subtypes are potent suppressors of T cell immunity, and their contribution has been investigated in a plethora of diseases including renal cancer, renal transplant, and chronic kidney disease. Whether MDSCs contribute to the pathogenesis of acute kidney injury (AKI) remains unknown. Herein, using human C-reactive protein (CRP) transgenic (CRPtg) and CRP-deficient mice (CRP(-/-)) subjected to bilateral renal ischemia-reperfusion injury (IRI), we confirm our earlier finding that CRP exacerbates renal IRI and show for the first time that this effect is accompanied in CRPtg mice by a shift in the balance of kidney-infiltrating MDSCs toward a suppressive Ly6G(+)Ly6C(low) g-MDSC subtype. In CRPtg mice, direct depletion of g-MDSCs (using an anti-Gr1 monoclonal antibody) reduced the albuminuria caused by renal IRI, confirming they play a deleterious role. Remarkably, treatment of CRPtg mice with an antisense oligonucleotide that specifically blocks the human CRP acute-phase response also led to a reduction in renal g-MDSC numbers and improved albuminuria after renal IRI. Our study in CRPtg mice provides new evidence that MDSCs participate in the pathogenesis of renal IRI and shows that their pharmacological depletion is beneficial. If ongoing investigations confirm that CRP is an endogenous regulator of MDSCs in CRPtg mice, and if this action is recapitulated in humans, then targeting CRP or/and MDSCs might offer a new approach for the treatment of AKI.

Human C-reactive protein impedes entry of leptin into the CNS and attenuates its physiological actions in the CNS.

Defective central leptin signalling and impaired leptin entry into the CNS (central nervous system) represent two important aspects of leptin resistance in obesity. In the present study, we tested whether circulating human CRP (C-reactive protein) not only diminishes signalling of leptin within the CNS, but also impedes this adipokine's access to the CNS. Peripheral infusion of human CRP together with co-infused human leptin was associated with significantly decreased leptin content in the CSF of ob/ob mice. Furthermore, following peripheral infusion of human leptin, the CSF (cerebrospinal fluid) concentration of leptin in transgenic mice overexpressing human CRP was sharply lower than that achieved in similarly infused wild-type mice. Administration of LPS (lipopolysaccharide) to human CRP-transgenic mice dramatically elevated the concentrations of human CRP in the CSF. The i.c.v. (intracerebroventricular) delivery of human CRP into the lateral ventricles of ob/ob mice blocked the satiety and weight-reducing actions of human leptin, but not those of mouse leptin. I.c.v. injection of human CRP abolished hypothalamic signalling by human leptin, and ameliorated the effects of leptin on the expression of NPY (neuropeptide Y), AgRP (Agouti-related protein), POMC (pro-opiomelanocortin) and SOCS-3 (suppressor of cytokine signalling 3). Human CRP can impede the access of leptin to the CNS, and elevation of human CRP within the CNS can have a negative impact on the physiological actions of leptin.

Hepatic but Not CNS-Expressed Human C-Reactive Protein Inhibits Experimental Autoimmune Encephalomyelitis in Transgenic Mice.

We recently demonstrated that human C-reactive protein (CRP), expressed hepatically in transgenic mice (CRPtg), improved the outcome of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The liver is the primary site of CRP synthesis in humans and in CRPtg mice but is also expressed by both at low levels in the CNS. To determine if CNS expression of human CRP is sufficient to impact EAE, we generated neuronal CRP transgenic mice (nCRPtg) wherein human CRP expression is driven by the neuron-specific Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) gene promoter. We found that hepatically expressed/blood-borne CRP, but not CNS expressed CRP, lessened EAE severity. These outcomes indicate that the protective actions of human CRP in EAE are manifested in the periphery and not in the CNS and reveal a previously unappreciated site specificity for the beneficial actions of CRP in CNS disease.

Effects of the Dietary ω3:ω6 Fatty Acid Ratio on Body Fat and Inflammation in Zebrafish (Danio rerio).

The diets of populations in industrialized nations have shifted to dramatically increased consumption of ω6 polyunsaturated fatty acids (PUFA), with a corresponding decrease in the consumption of ω3 PUFA. This dietary shift may be related to observed increases in obesity, chronic inflammation, and comorbidities in the human population. We examined the effects of ω3:ω6 fatty acid ratios in the context of constant total dietary lipid on the growth, total body fat, and responses of key inflammatory markers in adult zebrafish (Danio rerio). Zebrafish were fed diets in which the ω3:ω6 PUFA ratios were representative of those in a purported ancestral diet (1:2) and more contemporary Western diets (1:5 and 1:8). After 5 mo, weight gain (fat free mass) of zebrafish was highest for those that received the 1:8 ratio treatment, but total body fat was lowest at this ratio. Measured by quantitative real-time RT-PCR, mRNA levels from liver samples of 3 chronic inflammatory response genes (C-reactive protein, serum amyloid A, and vitellogenin) were lowest at the 1:8 ratio. These data provide evidence of the ability to alter zebrafish growth and body composition through the quality of dietary lipid and support the application of this model to investigations of human health and disease related to fat metabolism.

C-reactive protein directly suppresses Th1 cell differentiation and alleviates experimental autoimmune encephalomyelitis.

Human C-reactive protein (CRP) is a serum-soluble pattern recognition receptor that serves as a marker of inflammation and directly contributes to innate immunity. In this study, we show that human CRP also directly contributes to adaptive immunity, that is, native CRP binds specifically to human Jurkat T cells and to mouse naive CD4(+) T cells and modulates their Th1 and Th2 responses. In vitro both exogenously added (purified) and endogenously expressed (via transfection) human CRP inhibited Th1 differentiation and augmented Th2 differentiation of naive CD4(+) T cells. In vivo for human CRP transgenic compared with wild-type mice, a lesser proportion of the T cells recovered from the spleens of healthy animals were Th1 cells. Moreover, in both CRP transgenic mice and in wild-type mice treated with human CRP, during myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis both the Th1 cell response and disease severity were inhibited. These pattern recognition-independent actions of CRP directly on T cells highlights the potential for this soluble pattern recognition receptor to act as a tonic regulator of immunity, shaping global adaptive immune responses during both homeostasis and disease.

Human neutrophil flow chamber adhesion assay.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the ß2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both ß2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

Deletion of C-reactive protein ameliorates experimental cerebral malaria?

BACKGROUND: C-reactive protein (CRP) level correlates with parasitemia and severity of malaria, but whether this reflects causality remains unknown. METHODS: Using CRP-transgenic and CRP-deficient mice we compared the onset and severity of experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA). RESULTS: CRP-deficient mice were most resistant to ECM. CONCLUSIONS: CRP might contribute to the development of cerebral malaria, rather than protect against it.

Associations between abnormal rod-mediated dark adaptation and health and functioning in older adults with normal macular health.

PURPOSE: Delayed rod-mediated dark adaptation (DA) is characteristic of early age-related macular degeneration (AMD) and also can be observed in some older adults in normal macular health. We examine cross-sectional associations between rod-mediated DA and risk factors for AMD in older adults in normal macular health. METHODS: The sample consisted of adults aged ≥60 years old in normal macular health per grading of fundus photos using an established disease classification system. Rod-mediated DA was measured psychophysically following a photobleach using a computer-automated dark adaptometer with targets centered at 5° on the inferior vertical meridian. The speed of DA was characterized by the rod-intercept value, with abnormal DA defined as rod-intercept ≥ 12.3 minutes. We assessed several health and functional characteristics that the literature has suggested increase AMD risk (e.g., smoking, alcohol use, inflammatory markers, apolipoproteins, low luminance visual acuity, chronic medical conditions, body mass, family history). RESULTS: Among 381 participants (mean age, 68.5 years; SD, 5.5), 78% had normal and 22% had abnormal DA, with the prevalence of abnormal DA increasing with age. After age-adjustment, abnormal DA was associated with increased odds of elevated C-reactive protein (CRP), heavy use of or abstention from alcohol, high blood pressure, and drop in visual acuity under mesopic conditions. CONCLUSIONS: Despite having normal macular health according to accepted definitions of AMD presence, approximately one-quarter of older adults recruited from primary eye care clinics had abnormal DA, which was associated with known risk factors for AMD, including elevated CRP.

Inhibiting C-reactive protein for the treatment of cardiovascular disease: promising evidence from rodent models.

Raised blood C-reactive protein (CRP) level is a predictor of cardiovascular events, but whether blood CRP is causal in the disease process is unknown. The latter would best be defined by pharmacological inhibition of the protein in the context of a randomized case-control study. However, no CRP specific drug is currently available so such a prospective study cannot be performed. Blood CRP is synthesized primarily in the liver and the liver is an organ where antisense oligonucleotide (ASO) drugs accumulate. Taking advantage of this we evaluated the efficacy of CRP specific ASOs in rodents with experimentally induced cardiovascular damage. Treating rats for 4 weeks with a rat CRP-specific ASO achieved >60% reduction of blood CRP. Notably, this effect was associated with improved heart function and pathology following myocardial infarction (induced by ligation of the left anterior descending artery). Likewise in human CRP transgenic mice treated for 2 weeks with a human CRP-specific ASO, blood human CRP was reduced by >70% and carotid artery patency was improved (2 weeks after surgical ligation). CRP specific ASOs might pave the way towards a placebo-controlled trial that could clarify the role of CRP in cardiovascular disease.

Estrogen effects on vascular inflammation are age dependent: role of estrogen receptors.

OBJECTIVE: 17ß-Estradiol (E2) offers cardiovascular protection in young female animals and postmenopausal women. In contrast, randomized trials of menopausal hormones performed in older women have shown harm or no cardiovascular benefit. We hypothesize that E2 effects on vascular inflammation are age dependent. APPROACH AND RESULTS: Young (10 weeks) and aged (52 weeks) female C57BL/6 mice were used as source for primary cultures of bone marrow-derived macrophages (BMMs) and vascular smooth muscle cells (VSMCs). E2 pretreatment of cells derived from young mice attenuated C-reactive protein (CRP)-induced expression of inflammatory mediators. In contrast, E2 pretreatment of cells from aged mice did not alter (BMMs) or paradoxically exaggerated (VSMCs) inflammatory mediator response to CRP. Using E2 receptor (ER) knockout mice, we demonstrated that E2 regulates inflammatory response to CRP in BMMs via ERα and in VSMCs via ERß. BMMs derived from aged (versus young) mice expressed significantly less ERα mRNA and protein. A selective ligand of the novel ER GPR30 reproduced the E2 effects in BMMs and VSMCs. Unlike in young mice, E2 did not reduce neointima formation in ligated carotid arteries of aged CRP transgenic mice. CONCLUSIONS: E2 attenuates inflammatory response to CRP in BMMs and VSMCs derived from young but not aged mice and reduces neointima formation in injured carotid arteries of young but not aged CRP transgenic mice. ERα expression in BMMs is greatly diminished with aging. These data suggest that vasoprotective effects of E2 are age dependent and may explain the vasotoxic effects of E2 seen in clinical trials of postmenopausal women.

C-reactive protein promotes acute kidney injury by impairing G1/S-dependent tubular epithelium cell regeneration.

CRP (C-reactive protein) is regarded as an inflammatory biomarker in AKI (acute kidney injury), but its exact role in AKI remains unclear. Thus we sought to investigate the role of CRP in AKI. Clinically, elevated serum CRP levels were found to associate closely with increased serum creatinine and urea levels (P<0.01) in patients with AKI, which then fell after recovery from AKI. To determine the role of CRP in AKI, an ischaemia/reperfusion mouse model of AKI was developed using Tg (transgenic) mice that express human CRP. Compared with the WT (wild-type) mice, CRP Tg mice developed more severe renal injury at 24 h after ischaemia as determined by significantly increased serum creatinine and tubular necrosis. This was associated with an impaired TEC (tubular epithelium cell) regeneration as shown by an over 60% reduction in PCNA+ (proliferating-cell nuclear antigen) and BrdU+ (bromodeoxyuridine) TECs in CRP Tg mice with AKI. In vitro, the addition of CRP to a human TEC line (HK-2) also largely suppressed the proliferation of TECs. The functional role of CRP in AKI was demonstrated further by the blocking of CRP binding to the FcγRII (Fcγ receptor II) with a neutralizing anti-CD32 antibody, which restored TEC proliferation and prevented AKI in CRP Tg mice. Moreover, we found that impaired G1/S transition by suppression of the phosphorylation of CDK2 (cyclin-dependent kinase 2) and expression of cyclin E may be a key mechanism by which CRP inhibits TEC regeneration during the AKI repair process. In conclusion, CRP plays a pathogenic role in AKI by inhibiting G1/S-dependent TEC regeneration. The results of the present study suggest that targeting CRP signalling may offer a new therapeutic potential for AKI.