B Cell,Th17, and Neutrophil Related Cerebrospinal Fluid Cytokine/Chemokines Are Elevated in MOG Antibody Associated Demyelination

Background

Myelin oligodendrocyte glycoprotein antibody (MOG Ab) associated demyelination represents a subgroup of autoimmune demyelination that is separate from multiple sclerosis and aquaporin 4 IgG-positive NMO, and can have a relapsing course. Unlike NMO and MS, there is a paucity of literature on immunopathology and CSF cytokine/chemokines in MOG Ab associated demyelination.

Aim

To study the differences in immunopathogenesis based on cytokine/chemokine profile in MOG Ab-positive (POS) and -negative (NEG) groups.

Methods

We measured 34 cytokines/chemokines using multiplex immunoassay in CSF collected from paediatric patients with serum MOG Ab POS [acute disseminated encephalomyelitis (ADEM = 8), transverse myelitis (TM = 2) n = 10] and serum MOG Ab NEG (ADEM = 5, TM = 4, n = 9) demyelination. We generated normative data using CSF from 20 non-inflammatory neurological controls.

Results

The CSF cytokine and chemokine levels were higher in both MOG Ab POS and MOG Ab NEG demyelination groups compared to controls. The CSF in MOG Ab POS patients showed predominant elevation of B cell related cytokines/chemokines (CXCL13, APRIL, BAFF and CCL19) as well as some of Th17 related cytokines (IL-6 AND G-CSF) compared to MOG Ab NEG group (all p<0.01). In addition, patients with elevated CSF MOG antibodies had higher CSF CXCL13, CXCL12, CCL19, IL-17A and G-CSF than patients without CSF MOG antibodies.

Conclusion

Our findings suggest that MOG Ab POS patients have a more pronounced CNS inflammatory response with elevation of predominant humoral associated cytokines/chemokines, as well as some Th 17 and neutrophil related cytokines/chemokines suggesting a differential inflammatory pathogenesis associated with MOG antibody seropositivity. This cytokine/chemokine profiling provides new insight into disease pathogenesis, and improves our ability to monitor inflammation and response to treatment. In addition, some of these molecules may represent potential immunomodulatory targets.     

A Bioplex Analysis of Cytokines and Chemokines in First Trimester Maternal Plasma to Screen for Predictors of Miscarriage

Background

We have previously shown in two independent cohorts that circulating first trimester Macrophage Inhibitory Cytokine-1 (MIC-1) levels are lower in women in early pregnancy who are destined to miscarriage. While promising, the diagnostic performance of measuring MIC-1 alone was not sufficient for it to be a useful predictive test for miscarriage. Besides MIC-1, there are other cytokines, as well as chemokines, involved in facilitating early pregnancy. We reasoned that screening these factors in maternal plasma could uncover other predictive markers of miscarriage.

Methods

This was a nested case control study, of 78 women from a prospective study of 462 attending the Early Pregnancy Assessment Unit in the first trimester (EPAU) with a threatened miscarriage; 34 of these subsequently miscarried (cases) and 44 went on to have a normal delivery (controls) Cytokines IL-1β, IL-6 and IL-10, and the chemokines, CXCL8, CCL2, CCL5, CCL7 and CX3CL1 were measured in plasma from our cohort.

Results

The cytokines IL-1β, IL-6, IL-10 and the chemokine CXCL8 were not detectable in first trimester plasma. The chemokines CCL2, CCL5, CCL7 and CX3CL1 were detectable in all samples but levels did not vary across 5–12 weeks of gestation among controls. Plasma levels of these chemokines were no different in the miscarriage cohort compared to controls.

Conclusion

The chemokines CCL2, CCL5, CCL7 and CX3CL1 were detectable in plasma during the first trimester while IL-1β, IL-6, IL-10 and CXCL8 were not. However, none of the cytokines and chemokines screened were different in maternal plasma in cases or controls. These therefore do not appear to have potential for application as predictive biomarkers of miscarriage.     

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.     

Activation of eosinophils interacting with dermal fibroblasts by pruritogenic cytokine IL-31 and alarmin IL-33: implications in atopic dermatitis

Background

IL-31 is a pruritogenic cytokine, and IL-33 is an alarmin for damaging inflammation. They together relate to the pathogenesis of atopic dermatitis (AD). Eosinophil infiltration into the inner dermal compartment is a predominant pathological feature of AD. We herein investigated the in vitro inflammatory effects of IL-31 and IL-33 on the activation of human eosinophils and dermal fibroblasts.

Methodology/Principal Findings

Receptors, adhesion molecules and signaling molecules were assessed by Western blot or flow cytometry. Chemokines and cytokine were quantitated by multiplex assay. Functional IL-31 receptor component IL-31RA, OSMR-β and IL-33 receptor component ST2 were constitutively expressed on the surface of eosinophils. Co-culture of eosinophils and fibroblasts significantly induced pro-inflammatory cytokine IL-6 and AD-related chemokines CXCL1, CXCL10, CCL2 and CCL5. Such inductions were further enhanced with IL-31 and IL-33 stimulation. IL-31 and IL-33 could significantly provoke the release of CXCL8 from eosinophils and fibroblasts, respectively, which was further enhanced upon co-culture. In co-culture, eosinophils and fibroblasts were the main source for the release of CCL5, and IL-6, CXCL1, CXCL8, CXCL10 and CCL2, respectively. Direct interaction between eosinophils and fibroblasts was required for CXCL1, CXCL10, CXCL8 and CCL5 release. Cell surface expression of intercellular adhesion molecule-1 on eosinophils and fibroblasts was up-regulated in co-culture upon IL-31 and IL-33 stimulation. The interaction between eosinophils and fibroblasts under IL-31 and IL-33 stimulation differentially activated extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, nuclear factor-κB and phosphatidylinositol 3-kinase–Akt pathways. Using specific signaling molecule inhibitors, the differential induction of IL-31 and IL-33-mediated release of cytokines and chemokines such as IL-6 and CXCL8 from co-culture should be related to their distinct activation profile of intracellular signaling pathways.

Conclusions/Significance

The above findings suggest a crucial immunopathological role of IL-31 and IL-33 in AD through the activation of eosinophils-fibroblasts interaction via differential intracellular signaling mechanisms.     

Cerebrospinal fluid light and heavy neurofilaments in neuromyelitis optica

Background

Axonal injury is the correlate of disease progression in NMO and MS. Neurofilament (Nf) belongs to neuron specific intermediate filaments located in axons. Nf protein subunits are potential biomarkers in cerebrospinal fluid (CSF) for acute axonal injury. However, whether CSF NfH and NfL levels are elevated in NMO patients has remained unclear.

Methods

Nf light subunit (NfL) and Nf heavy subunit NfH in cerebrospinal fluid were measured by enzyme-linked immunosorbent assay (ELISA) in NMO (n = 32), MS (n = 25), and other non-inflammatory neurological disease patients (OND, n = 18).

Results

CSF pNf-H levels were increased in the NMO patients compared with OND patients (p = 0.001). CSF NfL levels in the NMO patients were also higher compared with MS patients (p = 0.001), and OND patients (p = 0.000001). When comparing NfL levels between MS and OND patients, there also significant differences (p = 0.0003). NMO and MS patients revealed a trend to an increased disability with increased CSF NfL during relapse (NMO: p = 0.006; MS: p = 0.017). There is positive relationship between CSF pNf-H and disability of MS patients (p = 0.041).

Conclusions

CSF levels of NfL are increased in NMO patients and reflect the disease severity in NMO.     

Feasibility of the Use of Combinatorial Chemokine Arrays to Study Blood and CSF in Multiple Sclerosis

Meningeal inflammation, including the presence of semi-organized tertiary lymphoid tissue, has been associated with cortical pathology at autopsy in secondary progressive multiple sclerosis (SPMS).  Accessible and robust biochemical markers of cortical inflammation for use in SPMS clinical trials are needed.  Increased levels of chemokines in the cerebrospinal fluid (CSF) can report on inflammatory processes occurring in the cerebral cortex of MS patients.  A multiplexed chemokine array that included BAFF, a high sensitivity CXCL13 assay and composite chemokine scores were developed to explore differences in lymphoid (CXCL12, CXCL13, CCL19 and CCL21) and inflammatory (CCL2, CXCL9, CXCL10 and CXCL11) chemokines in a small pilot study.  Paired CSF and serum samples were obtained from healthy controls (n=12), relapsing-remitting MS (RRMS) (n=21) and SPMS (N=12). A subset of the RRMS patients (n = 9) was assessed upon disease exacerbation and 1 month later following iv methylprednisone. SPMS patients were sampled twice to ascertain stability. Both lymphoid and inflammatory chemokines were elevated in RRMS and SPMS with the highest levels found in the active RRMS group. Inflammatory and lymphoid chemokine signatures were defined and generally correlated with each other. This small exploratory clinical study shows the feasibility of measuring complex and potentially more robust chemokine signatures in the CSF of MS patients during clinical trials. No differences were found between stable RRMS and SPMS. Future trials with larger patient cohorts with this chemokine array are needed to further characterize the differences, or the lack thereof, between stable RRMS and SPMS.        

Biomarkers of Inflammation and Axonal Degeneration/Damage in Patients with Newly Diagnosed Multiple Sclerosis: Contributions of the Soluble CD163 CSF/Serum Ratio to a Biomarker Panel

Background

Expression of soluble CD163 (sCD163), a macrophage/microglia biomarker, is increased in inflammatory conditions, and sCD163 levels in the cerebrospinal fluid (CSF) have recently been shown to be elevated in patients with multiple sclerosis (MS): the sCD163 CSF/serum ratio was elevated in patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and clinically isolated syndrome (CIS) compared with symptomatic controls.

Objective

To investigate the contributions of the sCD163 CSF/serum ratio to a biomarker panel focusing on inflammation and axonal degeneration in newly diagnosed MS; thus optimising a diagnostic biomarker panel for MS.

Methods

After a full MS diagnostic work-up, including collection of paired samples of CSF and serum, 125 patients were included in this study. Patients were divided into groups based on their diagnosis, and patients with normal clinical and paraclinical findings were defined as symptomatic controls. Serum and CSF levels, ratios, and indices of sCD163, CXCL13, osteopontin, neopterin, and CSF levels of neurofilament light polypeptide were determined by enzyme-linked immunosorbent assays (ELISAs). For sCD163 the results constitute a post-hoc analysis of already published data.

Results

All tested biomarkers, notably the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the CSF level of NfL, the IgG index, and the serum level of OPN, were significantly correlated to RRMS, PPMS, and/or CIS. The individual biomarkers in single tests had a lower performance than the IgG index, however, their combined receiver operating characteristic (ROC) curve demonstrated excellent diagnostic discriminatory power.

Conclusion

The biomarker panel showed distinct profiles for each patient group and could be a valuable tool for clinical differentiation of MS subgroups. The combined ROC analysis showed that sCD163 contributes positively as a diagnostic marker to a panel of established MS biomarkers. Patients with PPMS were demonstrated to have significantly elevated levels of both inflammatory and degenerative markers.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

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     

IL-17RA signaling amplifies antibody-induced arthritis

Objective

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

Methods

Wild-type and Il17ra^−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

Results

Il17ra^−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra^−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra^−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

Conclusions

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.     

Toll like Receptor 3 & 4 Responses of Human Turbinate Derived Mesenchymal Stem Cells: Stimulation by Double Stranded RNA and Lipopolysaccharide

Background and objectives

Multipotent mesenchymal stromal cells (MSCs) represent a promising cell-based therapy for a number of inflammatory or autoimmune diseases. Herein, Toll like receptor (TLR) expression by MSCs and their immune regulatory roles are investigated. In this study, we investigated the influence of TLR on the immune response, proliferation, and differentiation potential of human turbinated MSC (hTMSC) cultures in vitro.

Subjects and Methods

After isolating hTMSCs from discarded inferior turbinate tissue, FACS analysis was used to assess the expression of TLRs such as TLR2, TLR3, TLR4, and TLR5 in hTMSCs and cell proliferation was assessed using a cell counting kit (CCK)-8. Cytokine and chemokine secretions were analyzed with multiplex immunoassays for IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-a, GM-CSF, and IFN-γ. The differentiation potential of hTMSCs was evaluated in the osteogenic, chondogenic, and adipogeinc media and analyzed by histology and gene expression related to differentiation.

Results

FACS analysis revealed that TLR3 and TLR4 expression consisted of a relatively high percentage of the surface proteins expressed by hTMSCs. The proliferation of hTMSCs was influenced and significantly increased by the presence of TLR4 agonists. In particular, hTMSCs produced a set of cytokines and chemokines and the expression of IL-6, IL-8, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, and GM-CSF were up-regulated in response to the TLR4 agonist LPS. The osteogenic and adipogeinc differentiation potential of hTMSCs was not affected by TLR agonists.

Conclusions

We conclude that TLR4 stimulation affects TLR expression, proliferation, and the immunomodulation potential of hTMSCs. Understanding the mechanism behind TLR''s influence on hTMSCs and their immunomodulating properties would be useful for providing a novel target to exploit in the improvement of stem cell-based therapeutic strategies.     

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines

Background

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/Principal Findings

Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/Significance

The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.     

Elevated interleukin-12 and CXCL10 in subacute sclerosing panencephalitis

Immunosuppression associated with measles virus (MV) can be demonstrated by cytokine production failure in subacute sclerosing panencephalitis (SSPE) and may have implications on the pathogenesis of the disease. Cytokines (IL-12, IL-10, IL-4, IL-17, IL-18, IFN-alpha, IFN-gamma) and chemokines (CXCL8, CXCL10, CCL2 and CCL5) were measured in the cerebrospinal fluid (CSF) and serum samples from 60 patients with SSPE, 36 patients with infectious and/or inflammatory (IN) and 28 with other non-inflammatory (NIN) neurological diseases by ELISA. IL-12 p70+p40 was elevated in CSF and sera of SSPE when compared to the NIN group. However, the CSF levels of IL-12 p70 alone were not increased, indicating an increase of p40. The CSF of SSPE patients also showed relatively higher levels of IL-10 than that of the NIN group. CXCL10 levels in CSF were significantly higher in SSPE, whereas CXCL8 was increased in sera compared to NIN. No difference was detected in IFN-gamma, IFN-alpha, IL-17, IL-18, IL-4 or CCL2 and CCL5 levels. These results demonstrate that immune response against MV in SSPE may be impaired, although some T cell/Th1 inducing stimulations are present.     

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.     

Blockade of T Cell Contact-Activation of Human Monocytes by High-Density Lipoproteins Reveals a New Pattern of Cytokine and Inflammatory Genes

Background

Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact.

Methodology/Principal Findings

To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNγ, CCL2, CCL5, TNF, IL-1β, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1β, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E₂—both involved in inflammatory response—were triggered by T cell contact and inhibited in the presence of HDL.

Conclusions/Significance

These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.     

Macrophage Activation in Acute Exacerbation of Idiopathic Pulmonary Fibrosis

Background

Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) is a common cause of disease acceleration in IPF and has a major impact on mortality. The role of macrophage activation in AE of IPF has never been addressed before.

Methods

We evaluated BAL cell cytokine profiles and BAL differential cell counts in 71 IPF patients w/wo AE and in 20 healthy volunteers. Twelve patients suffered from AE at initial diagnosis while sixteen patients developed AE in the 24 months of follow-up. The levels of IL-1ra, CCL2, CCL17, CCL18, CCL22, TNF-α, IL-1β, CXCL1 and IL-8 spontaneously produced by BAL-cells were analysed by ELISA.

Results

In patients with AE, the percentage of BAL neutrophils was significantly increased compared to stable patients. We found an increase in the production rate of the pro-inflammatory cytokines CXCL1 and IL-8 combined with an increase in all tested M2 cytokines by BAL-cells. An increase in CCL18 levels and neutrophil counts during AE was observed in BAL cells from patients from whom serial lavages were obtained. Furthermore, high baseline levels of CCL18 production by BAL cells were significantly predictive for the development of future AE.

Conclusions

BAL cell cytokine production levels at acute exacerbation show up-regulation of pro-inflammatory as well as anti-inflammatory/ M2 cytokines. Our data suggest that AE in IPF is not an incidental event but rather driven by cellular mechanisms including M2 macrophage activation.     

Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.     

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.     

Role of the pregnancy-specific glycoprotein in regulation of the cytokine and chemokine profiles of intact mononuclear cells

The effect of human pregnancy-specific glycoprotein (PSG) on the cytokine and chemokine production in vitro by intact mononuclear cells was studied by the method of flow fluorimetry. PSG inhibited production of the proinflammatory cytokines IL-6, IL-8, IL-17, IFN-γ, and TNF-α and chemokines CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1; at the same time, PSG stimulated IL-12(p70) production. Simultaneously with increasing the VEGF level, PSG inhibited production of IL-9, IL-13, G-CSF, and GM-CSF. The PSG effect discovered can be interpreted as a contribution into the immune tolerance formation during pregnancy.