S100A9 is a biliary protein marker of disease activity in primary sclerosing cholangitis

Background and Aims

Bile analysis has the potential to serve as a surrogate marker for inflammatory and neoplastic disorders of the biliary epithelium and may provide insight into biliary pathophysiology and possible diagnostic markers. We aimed to identify biliary protein markers of patients with primary sclerosing cholangitis (PSC) by a proteomic approach.

Methods

Bile duct-derived bile samples were collected from PSC patients (n = 45) or patients with choledocholithiasis (n = 24, the control group). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to analyse the proteins, 2-D-gel patterns were compared by densitometry, and brush cytology specimens were analysed by RT-PCR.

Results

A reference bile-duct bile proteome was established in the control group without signs of inflammation or maligancy comprising a total of 379 non-redundant biliary proteins; 21% were of unknown function and 24% had been previously described in serum. In PSC patients, the biliary S100A9 expression was elevated 95-fold (p<0.005), serum protein expression was decreased, and pancreatic enzyme expression was unchanged compared to controls. The S100A9 expression was 2-fold higher in PSC patients with high disease activity than in those with low activity (p<0.05). The brush cytology specimens from the PSC patients with high disease activity showed marked inflammatory activity and leukocyte infiltration compared to the patients with low activity, which correlated with S100A9 mRNA expression (p<0.05).

Conclusions

The bile-duct bile proteome is complex and its analysis might enhance the understanding of cholestatic liver disease. Biliary S100A9 levels may be a useful marker for PSC activity, and its implication in inflammation and carcinogenesis warrants further investigation.     

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

Background

The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development.

Methodology/Principal Findings

We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping.

Conclusions/Significance

We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.     

CXCR7 Functions as a Scavenger for CXCL12 and CXCL11

Background

CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.

Methodology/Principal Findings

We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.

Conclusions/Significance

The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.     

Lack of chemokine signaling through CXCR5 causes increased mortality, ventricular dilatation and deranged matrix during cardiac pressure overload

Rationale

Inflammatory mechanisms have been suggested to play a role in the development of heart failure (HF), but a role for chemokines is largely unknown. Based on their role in inflammation and matrix remodeling in other tissues, we hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling during HF.

Objective

We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5 in cardiac pathophysiology leading to HF.

Methods and Results

Mice harboring a systemic knockout of the CXCR5 (CXCR5^−/−) displayed increased mortality during a follow-up of 80 days after aortic banding (AB). Following three weeks of AB, CXCR5^−/− developed significant left ventricular (LV) dilatation compared to wild type (WT) mice. Microarray analysis revealed altered expression of several small leucine-rich proteoglycans (SLRPs) that bind to collagen and modulate fibril assembly. Protein levels of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced in AB CXCR5^−/− compared to AB WT mice. Electron microscopy revealed loosely packed extracellular matrix with individual collagen fibers and small networks of proteoglycans in AB CXCR5^−/− mice. Addition of CXCL13 to cultured cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF, we observed increased myocardial levels of CXCR5 and SLRPs, which was reversed following LV assist device treatment.

Conclusions

Lack of CXCR5 leads to LV dilatation and increased mortality during pressure overload, possibly via lack of an increase in SLRPs. This study demonstrates a critical role of the chemokine CXCL13 and CXCR5 in survival and maintaining of cardiac structure upon pressure overload, by regulating proteoglycans essential for correct collagen assembly.     

HER2-positive circulating tumor cells in breast cancer

Purpose

Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging.

Methods

Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®.

Results

Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03).

Conclusion

HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.     

CXCL12/CXCR4 Axis Triggers the Activation of EGF Receptor and ERK Signaling Pathway in CsA-Induced Proliferation of Human Trophoblast Cells

Introduction

Our previous study has demonstrated Cyclosporin A (CsA) promotes the proliferation of human trophoblast cells. Therefore, we further investigate the intracellular signaling pathway involved in the CsA-induced proliferation of human trophoblast cells.

Methods

Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the regulation of CsA on CXCL12 secretion in human trophoblast cells. Immunofluorescence analysis and western blotting analysis were used to investigate the role of CXCL12/CXCR4 axis in the CsA-induced epidermal growth factor receptor (EGFR) phosphorylation in human trophoblast cells. 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay was performed to analyze the involvement of EGFR and its downstream extracellular signal-regulated protein kinase (ERK) signaling pathway in the CsA-induced proliferation of human trophoblast cells.

Results

Low concentration of CsA promoted the secretion of CXCL12, and recombinant human CXCL12 promoted the phosphorylation of EGFR in primary human trophoblast cells and choriocarcinoma cell line JEG-3. The inhibition of CXCL12 or CXCR4 by either neutralizing antibodies or small interfering RNA (siRNA) could completely block the CsA-induced EGFR phosphorylation. The CsA-induced proliferation of human trophoblast cells was effectively abrogated by the EGFR inhibitor AG1478 as well as the ERK inhibitor U0126, but not by the PI3K/PKB inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. CsA promoted the activation of ERK in JEG-3 cells, which was markedly abrogated in the presence of CXCL12 siRNA, or CXCR4 siRNA, or AG1478.

Conclusions

CsA may promote EGFR activation via CXCL12/CXCR4 axis, and EGFR downstream ERK signaling pathway may be involved in the CsA-induced proliferation of human trophoblast cells.     

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis

Background

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

Methodology

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

Principal Findings

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

Conclusions

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.     

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

Background

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods

Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and ≤5 WBC/µL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/µL) patients. The concentration of H-FABP, GSTP-1 and S100β in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1β, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-γ, TNF-α, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results

CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion

This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.     

Effectiveness of carboplatin and paclitaxel as first- and second-line treatment in 61 patients with metastatic melanoma

Background

Patients with metastatic melanoma have a very unfavorable prognosis with few therapeutic options. Based on previous promising experiences within a clinical trial involving carboplatin and paclitaxel a series of advanced metastatic melanoma patients were treated with this combination.

Methods

Data of all patients with cutaneous metastatic melanoma treated with carboplatin and paclitaxel (CP) at our institution between October 2005 and December 2007 were retrospectively evaluated. For all patients a once-every-3-weeks dose-intensified regimen was used. Overall and progression free survival were calculated using the method of Kaplan and Meier. Tumour response was evaluated according to RECIST criteria.

Results

61 patients with cutaneous metastatic melanoma were treated with CP. 20 patients (85% M1c) received CP as first-line treatment, 41 patients (90.2% M1c) had received at least one prior systemic therapy for metastatic disease. Main toxicities were myelosuppression, fatigue and peripheral neuropathy. Partial responses were noted in 4.9% of patients, stable disease in 23% of patients. No complete response was observed. Median progression free survival was 10 weeks. Median overall survival was 31 weeks. Response, progression-free and overall survival were equivalent in first- and second-line patients. 60 patients of 61 died after a median follow up of 7 months. Median overall survival differed for patients with controlled disease (PR+SD) (49 weeks) compared to patients with progressive disease (18 weeks).

Conclusions

Among patients with metastatic melanoma a subgroup achieved disease control under CP therapy which may be associated with a survival benefit. This potential advantage has to be weighed against considerable toxicity. Since response rates and survival were not improved in previously untreated patients compared to pretreated patients, CP should thus not be applied as first-line treatment.     

Atorvastatin Reduces Plasma Levels of Chemokine (CXCL10) in Patients with Crohn's Disease

Background

In Crohn''s disease high tissue expression and serum levels of chemokines and their receptors are known to correlate with disease activity. Because statins can reduce chemokine expression in patients with coronary diseases, we wanted to test whether this can be achieved in patients with Crohn''s disease.

Methodology/Principal Findings

We investigated plasma levels of chemokines (CCL2, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8, CXCL10) and endothelial cytokines (sP-selectin, sE-selectin, sICAM-3, thrombomodulin) in ten Crohn''s disease patients before and after thirteen weeks'' daily treatment with 80 mg atorvastatin. Of the 13 substances investigated, only CXCL10 was found to be significantly reduced (by 34%, p = 0.026) in all of the treated patients. Levels of CXCL10 correlated with C-reactive protein (r = 0.82, p<0.01).

Conclusions/Significance

CXCL10 is a ligand for the CXCR3 receptor, the activation of which results in the recruitment of T lymphocytes and the perpetuation of mucosal inflammation. Hence the reduction of plasma CXCL10 levels by atorvastatin may represent a candidate for an approach to the treatment of Crohns disease in the future.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00454545","term_id":"NCT00454545"}}NCT00454545     

Cannabinoids reduce markers of inflammation and fibrosis in pancreatic stellate cells

Background

While cannabinoids have been shown to ameliorate liver fibrosis, their effects in chronic pancreatitis and on pancreatic stellate cells (PSC) are unknown.

Methodology/Principal Findings

The activity of the endocannabinoid system was evaluated in human chronic pancreatitis (CP) tissues. In vitro, effects of blockade and activation of cannabinoid receptors on pancreatic stellate cells were characterized. In CP, cannabinoid receptors were detected predominantly in areas with inflammatory changes, stellate cells and nerves. Levels of endocannabinoids were decreased compared with normal pancreas. Cannabinoid-receptor-1 antagonism effectuated a small PSC phenotype and a trend toward increased invasiveness. Activation of cannabinoid receptors, however, induced de-activation of PSC and dose-dependently inhibited growth and decreased IL-6 and MCP-1 secretion as well as fibronectin, collagen1 and alphaSMA levels. De-activation of PSC was partially reversible using a combination of cannabinoid-receptor-1 and -2 antagonists. Concomitantly, cannabinoid receptor activation specifically decreased invasiveness of PSC, MMP-2 secretion and led to changes in PSC phenotype accompanied by a reduction of intracellular stress fibres.

Conclusions/Significance

Augmentation of the endocannabinoid system via exogenously administered cannabinoid receptor agonists specifically induces a functionally and metabolically quiescent pancreatic stellate cell phenotype and may thus constitute an option to treat inflammation and fibrosis in chronic pancreatitis.     

Microfluidic Endothelium for Studying the Intravascular Adhesion of Metastatic Breast Cancer Cells

Background

The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation.

Methodology/Principal Findings

We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.

Conclusions/Significance

When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.     

CXCL1 Contributes to β-Amyloid-Induced Transendothelial Migration of Monocytes in Alzheimer’s Disease

Background

Bone marrow-derived microglia that originates in part from hematopoietic cells, and more particularly from monocytes preferentially attach to amyloid deposition in brains of Alzheimer’s disease (AD). However, the mechanism of monocytes recruited into the amyloid plaques with an accelerated process in AD is unclear.

Methodology/Principal Findings

Here we reported that monocytes from AD patients express significantly higher chemokine (C-X-C motif) ligand 1 (CXCL1) compared to age-matched controls. AD patient’s monocytes or CXCL1-overexpressing THP-1 cells had enhanced ability of β-amyloid (Aβ)-induced transendothelial migration and Aβ-induced transendothelial migration for AD patient’s monocytes or CXCL1-overexpressing THP-1 cells was almost abrogated by anti-CXCL1 antibody. Furthermore, monocytes derived from a transgenic mouse model of AD also expressed significantly higher CXCL1. CD11b⁺CD45^hi population of cells that were recruited from the peripheral blood were markedly bolcked in APP mouse brain by anti-CXCL1 antibody. Accordingly, in response to Aβ, human brain microvascular endothelial cells (HBMEC) significantly up-regulated CXC chemokine receptor 2 (CXCR2) expression, which was the only identified receptor for CXCL1. In addition, a high level expression of CXCR2 in HBMEC significantly promoted the CXCL1-overexpressing THP-1 cells transendothelial migration, which could be was abrogated by anti-CXCR2 antibody. Further examination of possible mechanisms found that CXCL1-overexpressing THP-1 cells induced transendothelial electrical resistance decrease, horseradish peroxidase flux increase, ZO-1 discontinuous and occludin re-distribution from insoluble to soluble fraction through interacting with CXCR2. ROCK inhibitor, Y27632, could block CXCL1-overexpressing THP-1 cells transendothelial migration, whereas other inhibitors had no effects.

Conclusions/Significance

The present data indicate that monocytes derived from AD patients overexpressing CXCL1, which is a determinant for Aβ-induced transendothelial migration. CXCL1 expressed by monocytes and CXCR2 on HBMEC is involved in monocytes migrating from blood to brain in AD patients.     

Epigenetics underpinning the regulation of the CXC (ELR+) chemokines in non-small cell lung cancer

Background

Angiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR⁺) chemokine family are powerful promoters of the angiogenic response.

Methods

The expression of the CXC (ELR⁺) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines.

Results

Overall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line.

Conclusions

CXC (ELR⁺) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment.     

Cytokine response patterns in severe pandemic 2009 H1N1 and seasonal influenza among hospitalized adults

Background

Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis.

Methods

Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed.

Results

63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2–15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2–27 times lower than seasonal influenza; P−values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3–4, 55% vs 85%; day 6–7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6–61.5, per log₁₀unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearman''s rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their ‘responsiveness’ to stimuli was shown to change dynamically during the illness course.

Conclusions

A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis.