Inflammatory Soluble Factors Research Articles

Characteristic Profiling of Soluble Factors in the Cerebrospinal Fluid of Patients With Neurosyphilis.

Soluble inflammatory factors in the cerebrospinal fluid (CSF) of patients with neurosyphilis have been investigated with low-throughput technology. This study aimed to illustrate the characteristics of soluble factor profiles in CSF of patients with neurosyphilis. We measured the concentrations of 45 cytokines, chemokines, and growth factors in CSF from 112 untreated syphilis cases, including latent syphilis (LS), asymptomatic neurosyphilis (ANS), meningeal neurosyphilis (MNS), meningovascular neurosyphilis (MVNS), paralytic dementia (PD), and ocular syphilis (OS). Thirty-three differentially expressed soluble factors (DeSFs) were categorized into 3 clusters. DeSF scores of clusters 1 and 2 (DeSFS1 and DeSFS2) were positively correlated with elevated neopterin and neurofilament light subunit (NF-L) concentration, respectively. DeSF scores of cluster 3 were positively correlated with white blood cells, protein, NF-L, and neopterin. Patients with LS, ANS, and OS exhibited an overall lower abundance of DeSFs. Patients with PD exhibited significantly increased levels of clusters 1 and 3, and the highest total DeSF score, whereas patients with MNS and MVNS showed enhanced levels of cluster 2. Receiver operating characteristic analysis revealed that DeSFS1 effectively discriminated PD, and DeSFS2 discriminated MNS/MVNS with high accuracy. Patients with neurosyphilis at different stages have distinctive patterns of soluble factors in CSF, which are correlated with immune status and neuronal damage.

Bone Marrow Cytokines Concentrations in Myelodysplastic Neoplasms: Its Correlation with the Immune Populations and Clonal Hematopoiesis

Background: Myelodysplastic neoplasms (MDS) are a clonal, heterogenic disease of the hematopoietic stem cell. Pathophysiology is mainly related to intrinsic myeloid abnormalities, epigenetic changes and immune system deregulation that leads to changes in the bone marrow microenvironment. MDS patients have shown broad and heterogenic changes in cytokine concentrations compared to controls. An elevation of pro-apoptotic and inflammatory cytokines and chemokines has been described. Nevertheless, the role of inflammatory soluble factors is still poorly understood. We aim to describe cytokine concentrations in the bone marrow of MDS patients and how it correlates with the immune population and clonal hematopoiesis. Methods: We prospectively analyzed 60 bone marrow plasma samples. Forty-four MDS patients without RS or 5q deletion, 8 healthy donors (HD) and 9 cytopenia of unknown significance (CUS). Cytokine/chemokine concentration were analyzed with ThermoFisher commercial kit ProcartaPlexTM Multiplex immunoassay of 32-PLEX (EGF/eotaxin/FGF-2/G-CSF/GM-CSF/HGF/INFalpha/ IFNgamma/IL-1alpha,/IL1-b/IL-10/IL-12/IL-13/IL-15/IL-17a/IL-1RA/IL-2/IL-22 /IL3 /IL-4/IL-5/IL-6/IL-7/IL-8/IL-9), carried out by Luminex® platform. Furthermore, we analyzed NK subpopulations differentiating an immature population (CD56 brigthCD16 −) from a mature one (CD56 dimCD16+). Lymphocyte T sub-populations were studied in PB samples determined as CD45+CD3+ and the different expression of the following monoclonal antibodies CD4/CD8/CCR7/CD45RA/CD27/CD28/CD25/CD127/CXCR3/CXCR6. The analysis was performed, by flow cytometry, on fresh samples, following a membrane staining protocol with 10-colors/3 lasers. Finally, molecular analysis was performed by NGS using the Oncomine Myeloid Research Assay included 40 genes. Results: A total of 60 subjects were included (median age was 71 years-old and 35% were female). Auto-immune disease was present in 6.6% (Figure 1). Regarding chemokines, CCL11 a pro-inflammatory chemokine was increased in MDS (59.73pg/ml) compared to CUS (41.24pg/ml) and HD (22.63pg/ml), also CXCL9, with a complex role in tumor progression, being 29.08pg/ml in MDS, 17.14pg/ml in CUS and 1.13pg/ml in HD. On the other hand, CCL2 and CCL4 showed lower concentrations in MDS vs HD but higher than CUS (Figure 2). Interleukins analysis showed, IL-1RA higher concentrations in MDS (139.9pg/ml), than HD (36.4pg/mL) and no detection in CUS. IL- 2 was detected only in HD (7.61pg/mL) and IL-3 showed greater concentrations in HD and CUS (20.75pg/ml) compared to MDS (13.84pg/ml). IL-6 did not showed differences among the three groups; in 18.3% subjects (11/60) concentrations were ≥10pg/ml and 8 were MDS. IL-8, a pro-inflammatory/proangiogenic cytokine, showed increased concentrations in only 23% subjects (14/60); 13 were MDS. Regarding growth factors, we observed EGF only in HD (28.16%), whilst HGF and VEGF-A showed higher concentrations in MDS. Mature NK sub-populations were increased in CUS (76.16%) vs MDS (11.58%), (p=0.006) and HD (10.66%), (p=0.01). Moreover, CD8+ T lymphocytes between CUS 49.79% vs MDS 14.29% and HD 1.68 (p=0.05) and Th2 in MDS of 22.71% vs CUS 17.5 (p=0.031) without differences to HD 22.54%. Forty-six MDS subjects presented ≥3 somatic mutations, those presented higher concentrations of CCL11 (73.56pg/mL), HGF (497.7pg/mL), CCL2 (95.3pg/ml) and VEGF-A (506pg/mL). Interestingly, we found that among CUS subjects 66.6% (6/9) presented mutations, 2 DMT3A (VAF < 5%), 1 IDH1 (VAF 3.7%), 1 SRSF2 (VAF 38.9%), 1 TET2 (VAF 39.5%), 1 ZRSR2 (VAF 35%), 1 PHF6 (VAF 39.8%). Finally, regarding IPSS-R risk, we observed higher concentrations of inhibitory proteins like IL-10 and cytokines involved in the regulation of tumor microenvironment like CXCL10 or CCL2 in HR-MDS, while in LR-MDS we observed a more cytotoxic and inflammatory profile (Figure 2). Conclusions: Our study describes a heterogenic background of MDS. Suggesting an increase in some cytokines related to cell recruitment, migration, and proliferation in the bone marrow of MDS and an exhaustive and inhibitory environment in those patients with high-risk characteristics, pointing out the important role of immune subpopulations and environment in the clonal progression.

Inflammatory profile of convalescent plasma to treat COVID: Impact of amotosalen/UVA pathogen reduction technology.

Blood products in therapeutic transfusion are now commonly acknowledged to contain biologically active constituents during the processes of preparation. In the midst of a worldwide COVID-19 pandemic, preliminary evidence suggests that convalescent plasma may lessen the severity of COVID-19 if administered early in the disease, particularly in patients with profound B-cell lymphopenia and prolonged COVID-19 symptoms. This study examined the influence of photochemical Pathogen Reduction Treatment (PRT) using amotosalen‐HCl and UVA light in comparison with untreated control convalescent plasma (n= 72 – paired samples) - cFFP, regarding soluble inflammatory factors: sCD40L, IFN-alpha, IFN-beta, IFN-gamma, IL-1 beta, IL-6, IL-8, IL-10, IL-18, TNF-alpha and ex-vivo inflammatory bioactivity on endothelial cells. We didn’t observe significant modulation of the majority of inflammatory soluble factors (8 of 10 molecules tested) pre- or post-PRT. We noted that IL-8 concentrations were significantly decreased in cFFP with PRT, whereas the IL-18 concentration was increased by PRT. In contrast, endothelial cell release of IL-6 was similar whether cFFP was pre-treated with or without PRT. Expression of CD54 and CD31 in the presence of cFFP were similar to control levels, and both were significant decreased in when cFFP had been pre-treated by PRT. It will be interesting to continue investigations of IL-18 and IL-8, and the physiopathological effect of PRT- treated convalescent plasma and in clinical trials. But overall, it appears that cFFP post-PRT were not excessively pro-inflammatory. Further research, including a careful clinical evaluation of CCP-treated patients, will be required to thoroughly define the clinical relevance of these findings.

S100A8/A9 Molecular Complexes Promote Cancer Migration and Invasion via the p38 MAPK Pathway in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is one type of malignancy associated with migration and invasion through a currently unclear mechanism. We previously discovered S100A8/A9 levels were roughly elevated in the plasma of NPC patients as the promising biomarkers. However, their expressions and underlying functions in NPC tissues are still unknown. In the present study, we analyzed 49 NPC tissues and 20 chronic pharyngitis (CP) tissues. Immunohistochemical staining was performed in different tissues and analyzed by the Mann–Whitney U test statistically. Transwell migration and invasion experiments were further performed to determine S100A8/A9 effects on NPC. Our results showed that S100A8/A9 in NPC tissues were significantly higher than those in CP tissues, closely associated with NPC clinical stages. Intriguingly, exogenous S100A8/A9 protein stimulation could dramatically enhance NPC migration and invasion abilities. In addition, p38 MAPK pathway blockade could diminish the migration and invasion of NPC cells stimulated by S100A8/A9 proteins. The downstream tumor invasion and migration associated proteins (e.g., MMP7) were also elevated in NPC tissues, consistent with S100A8/A9 overexpression. Taken together, our present findings suggest that the secreted soluble inflammatory factors S100A8/A9 might promote cancer migration and invasion via the p38 MAPK signaling pathway along with invasion/migration associated proteins overexpression in the tumor microenvironment of NPC. This may shed light on the mechanism understanding of NPC prognosis and provide more novel clues for NPC diagnosis and therapy.

The heterodimer S100A8/A9 is a potent therapeutic target for idiopathic pulmonary fibrosis.

In patients with interstitial pneumonia, pulmonary fibrosis is an irreversible condition that can cause respiratory failure. Novel treatments for pulmonary fibrosis are necessary. Inflammation is thought to activate lung fibroblasts, resulting in pulmonary fibrosis. Of the known inflammatory molecules, we have focused on S100A8/A9 from the onset of inflammation to the subsequent progression of inflammation. Our findings confirmed the high expression of S100A8/A9 in specimens from patients with pulmonary fibrosis. An active role of S100A8/A9 was demonstrated not only in the proliferation of fibroblasts but also in the fibroblasts' differentiation to myofibroblasts (the active form of fibroblasts). S100A8/A9 also forced fibroblasts to upregulate the production of collagen. These effects were induced via the receptor of S100A8/A9, i.e., the receptor for advanced glycation end products (RAGE), on fibroblasts. The anti-S100A8/A9 neutralizing antibody inhibited the effects of S100A8/A9 on fibroblasts and suppressed the progression of fibrosis in bleomycin (BLM)-induced pulmonary fibrosis mouse model. Our findings strongly suggest a crucial role of S100A8/A9 in pulmonary fibrosis and the usefulness of S100A8/A9-targeting therapy for fibrosis interstitial pneumonia. HIGHLIGHTS: S100A8/A9 level is highly upregulated in the IPF patients' lungs as well as the blood. S100A8/A9 promotes not only the growth of fibroblasts but also differentiation to myofibroblasts. The cell surface RAGE acts as a crucial receptor to the extracellular S100A8/A9 in fibroblasts. The anti-S100A8/A9 antibody effectively suppresses the progression of IPF in a mouse model. In idiopathic pulmonary fibrosis (IPF), S100A8/A9, a heterodimer composed of S100A8 and S100A9 proteins, plays a crucial role in the onset of inflammation and the subsequent formation of a feed-forward inflammatory loop that promotes fibrosis. (1) The local, pronounced increase in S100A8/A9 in the injured inflammatory lung region-which is provided mainly by the activated neutrophils and macrophages-exerts strong inflammatory signals accompanied by dozens of inflammatory soluble factors including cytokines, chemokines, and growth factors that further act to produce and secrete S100A8/A9, eventually making a sustainable inflammatory circuit that supplies an indefinite presence of S100A8/A9 in the extracellular space with a mal-increased level. (2) The elevated S100A8/A9 compels fibroblasts to activate through receptor for advanced glycation end products (RAGE), one of the major S100A8/A9 receptors, resulting in the activation of NFκB, leading to fibroblast mal-events (e.g., elevated cell proliferation and transdifferentiation to myofibroblasts) that actively produce not only inflammatory cytokines but also collagen matrices. (3) Finally, the S100A8/A9-derived activation of lung fibroblasts under a chronic inflammation state leads to fibrosis events and constantly worsens fibrosis in the lung. Taken together, these findings suggest that the extracellular S100A8/A9 heterodimer protein is a novel mainstay soluble factor for IPF that exerts many functions as described above (1-3). Against this background, we herein applied the developed S100A8/A9 neutralizing antibody to prevent IPF. The IPF imitating lung fibrosis in an IPF mouse model was effectively blocked by treatment with the antibody, leading to enhanced survival. The developed S100A8/A9 antibody, as an innovative novel biologic, may help shed light on the difficulties encountered with IPF therapy in clinical settings.

Intrathecal Inflammation in Progressive Multiple Sclerosis

Progressive forms of multiple sclerosis (MS) are associated with chronic demyelination, axonal loss, neurodegeneration, cortical and deep gray matter damage, and atrophy. These changes are strictly associated with compartmentalized sustained inflammation within the brain parenchyma, the leptomeninges, and the cerebrospinal fluid. In progressive MS, molecular mechanisms underlying active demyelination differ from processes that drive neurodegeneration at cortical and subcortical locations. The widespread pattern of neurodegeneration is consistent with mechanisms associated with the inflammatory molecular load of the cerebrospinal fluid. This is at variance with gray matter demyelination that typically occurs at focal subpial sites, in the proximity of ectopic meningeal lymphoid follicles. Accordingly, it is possible that variations in the extent and location of neurodegeneration may be accounted for by individual differences in CSF flow, and by the composition of soluble inflammatory factors and their clearance. In addition, “double hit” damage may occur at sites allowing a bidirectional exchange between interstitial fluid and CSF, such as the Virchow–Robin spaces and the periventricular ependymal barrier. An important aspect of CSF inflammation and deep gray matter damage in MS involves dysfunction of the blood–cerebrospinal fluid barrier and inflammation in the choroid plexus. Here, we provide a comprehensive review on the role of intrathecal inflammation compartmentalized to CNS and non-neural tissues in progressive MS.

Persistent Hyperactivation of Endothelial Cells in Patients with Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is a severe inflammatory liver disease that develops in some heavy drinkers. AH patients have intense hepatic infiltration of leukocytes. Up-regulation of cell adhesion molecules (CAMs) upon endothelial cell (EC) activation plays an important role in leukocyte transendothelial migration. CAMs can shed from EC surface and accumulate in the blood, serving as soluble markers for EC activation. In this study, we examined the impact of heavy drinking on expression of soluble forms of EC activation markers (CD146, ICAM-1, VCAM-1, and VEGF-A) and the effect of alcohol abstinence on the reversal of these abnormalities in heavy drinkerswith and without AH. ELISA and multiplex immunoassays were used to measure soluble EC activationmarkers in plasma samples from 79 AH patients, 66 heavy drinkers without overt liver disease (HDC), and 44 healthy controls (HC) at baseline, 31 AH patients and 30 HDC at 6-month follow-up, and 18 AH patients and 25 HDC at 12-month follow-up. At baseline, the 4 soluble markers were significantly up-regulated in AH patients compared with HDC and HC, whereas only sVCAM-1 was elevated in HDC relative to HC. At follow-ups, plasma levels of CD146, VCAM-1, and VEGF-A remained higher in AH patients, even for those who stopped drinking. These dysregulated markers correlated with AH disease severity, clinical parameters, and several solubleinflammatory factors. The levels of soluble CD146, ICAM-1, VCAM-1, and VEGF-A were highly elevated in AH patients, and alcohol abstinence did not completely reverse these abnormalities.

Identification of biomarkers for personalized peptide vaccination in 2,588 cancer patients.

Peptide-based cancer vaccines have failed to provide sufficient clinical benefits in order to be approved in clinical trials since the 1990s. To understand the mechanisms underlying this failure, the present study investigated biomarkers associated with the lower overall survival (OS) among 2,588 patients receiving personalized peptide vaccination (PPV). Survival data were obtained from a database of 2,588 cancer patients including 399 patients with lung, 354 with prostate and 344 with colon cancer. They entered into phase II clinical trials of PPV in which 2 to 4 of 31 warehouse peptides were selected for vaccination on an individual patient basis based on human leukocyte antigen (HLA) class IA-types and pre-existing peptide-specific IgG levels. Higher pre-vaccination neutrophil, monocyte and platelet counts, and lower pre-vaccination lymphocyte and red blood cell counts were inversely associated with OS, with higher sensitivities in the proportions of neutrophils and lymphocytes, respectively. The most potent unfavorable and favorable factors for OS were the median percentage of neutrophils (>64.8%) or percentage of lymphocytes (>25.1%) with correlation coefficients (R2) of 0.98 and 0.92, respectively. Higher pre-vaccination levels of c-reactive protein and other inflammatory soluble factors were inversely associated with OS. Pre-vaccination peptide-specific immunity levels had no effect on OS, although lower immune boosting levels were inversely associated with OS. None of the 31 peptides was inversely associated with OS, although a few peptides were positively associated with it. On the whole, the findings of the present study suggested that pre-vaccination inflammatory signatures, but not those of post-vaccination immune induction, were associated with lower clinical benefits of PPV.

Extracorporeal cytokine removal in severe CAR-T cell associated cytokine release syndrome

PurposeLife-threatening complications of CD-19 Chimeric antigen receptor - T (CAR-T) cells such as the cytokine release syndrome (CRS)) have been reported. Treatment is limited to IL-6 blockade and steroids although global removal of elevated soluble inflammatory factors might be more effective. MethodsClinical course of a CRS patient treated with extracorporeal cytokine adsorption (Cytosorb®). A panel of 48 cytokines, chemokines and endothelial markers has been analyzed longitudinally. Ex vivo stimulation of endothelial cells to visualize (immunocytochemistry) and quantify (ECIS, TER) endothelial barrier effects. ResultsFollowing CAR-T cell application a 65 years old male developed grade 4 CRS with refractory shock (3 vasopressors) and severe capillary leakage (+37 L/24 h resuscitation). Treatment included IL-6 blockade, methylprednisolone and additionally Cytosorb hemoperfusion. While multiple soluble inflammatory factors were elevated and most of them decreased by more than 50% following Cytosorb, markers of endothelial injury increased steadily (e.g. Angpt-2/Angpt-1) leading to profound endothelial activation and leakage in ex vivo assays. ConclusionThis is the first reported use of cytokine adsorption for CRS showing efficacy in absorption of various cytokines but not endothelial growth factors. A randomized controlled trial to evaluate additional Cytosorb treatment in CRS is currently recruiting at our institution (NCT04048434).

Overcoming immunotherapeutic resistance by targeting the cancer inflammation cycle.

Inflammation is a hallmark of cancer and supports tumor growth, proliferation, and metastasis, but also inhibits T cell immunosurveillance and the efficacy of immunotherapy. The biology of cancer inflammation is defined by a cycle of distinct immunological steps that begins during disease conception with the release of inflammatory soluble factors. These factors communicate with host organs to trigger bone marrow mobilization of myeloid cells, trafficking of myeloid cells to the tumor, and differentiation of myeloid cells within the tumor bed. Tumor-infiltrating myeloid cells then orchestrate an immunosuppressive microenvironment and assist in sustaining a vicious cycle of inflammation that co-evolves with tumor cells. This Cancer-Inflammation Cycle acts as a rheostat or "inflammostat" that impinges upon T cell immunosurveillance and prevents the development of productive anti-tumor immunity. Here, we define the major nodes of the Cancer-Inflammation Cycle and describe their impact on T cell immunosurveillance in cancer. Additionally, we discuss emerging pre-clinical and clinical data suggesting that intervening upon the Cancer-Inflammation Cycle will be a necessary step for broadening the potential of immunotherapy in cancer.

Lower Interferon Regulatory Factor-8 Expression in Peripheral Myeloid Cells Tracks With Adverse Central Nervous System Outcomes in Treated HIV Infection.

Cognitive dysfunction persists in 30–50% of chronically HIV-infected individuals despite combination antiretroviral therapy (ART). Although monocytes are implicated in poor cognitive performance, distinct biological mechanisms associated with cognitive dysfunction in HIV infection are unclear. We previously showed that a regulatory region of the interferon regulatory factor-8 (IRF8) gene is hyper-methylated in HIV-infected individuals with cognitive impairment compared to those with normal cognition. Here, we investigated IRF-8 protein expression and assessed relationships with multiple parameters associated with brain health. Intracellular IRF-8 expression was measured in cryopreserved peripheral blood mononuclear cells from chronically HIV-infected individuals on ART using flow cytometry. Neuropsychological performance was assessed by generating domain-specific standardized (NPZ) scores, with a global score defined by aggregating individual domain scores. Regional brain volumes were obtained by magnetic resonance imaging and soluble inflammatory factors were assessed by immunosorbent assays. Non-parametric analyses were conducted and statistical significance was defined as p < 0.05. Twenty aviremic (HIV RNA<50 copies/ml) participants, 84% male, median age 51 [interquartile range (IQR) 46, 55], median CD4 count 548 [439, 700] were evaluated. IRF-8 expression was highest in plasmacytoid dendritic cells (pDCs). Assessing cognitive function, lower IRF-8 density in classical monocytes significantly correlated with worse NPZ_learning memory (LM; rho = 0.556) and NPZ_working memory (WM; rho = 0.612) scores, in intermediate monocytes with worse NPZ_LM (rho = 0.532) scores, and in non-classical monocytes, lower IRF-8 correlated with worse global NPZ (rho = 0.646), NPZ_LM (rho = 0.536), NPZ_WM (rho = 0.647), and NPZ_executive function (rho = 0.605) scores. In myeloid DCs (mDCs) lower IRF-8 correlated with worse NPZ_WM (rho = 0.48) scores and in pDCs with worse NPZ_WM (rho = 0.561) scores. Declines in IRF-8 in classical monocytes significantly correlated with smaller hippocampal volume (rho = 0.573) and in intermediate and non-classical monocytes with smaller cerebral white matter volume (rho = 0.509 and rho = 0.473, respectively). IRF-8 density in DCs did not significantly correlate with brain volumes. Among biomarkers tested, higher soluble ICAM-1 levels significantly correlated with higher IRF-8 in all monocyte and DC subsets. These data may implicate IRF-8 as a novel transcription factor in the neuropathophysiology of brain abnormalities in treated HIV and serve as a potential therapeutic target to decrease the burden of cognitive dysfunction in this population.

The Interplay Between Systemic Inflammatory Factors and MicroRNAs in Age-Related Macular Degeneration.

We aimed to explore the expression of systemic inflammatory factors and selected intracellular miRNAs that regulate inflammatory signaling pathways potentially involved in age-related macular degeneration (AMD) pathogenesis. A total of 179 patients with wet AMD, 175 with dry AMD and 121 controls were enrolled in the study. Soluble inflammatory factors were analyzed in plasma samples using Luminex technology. Expression of selected miRNAs was analyzed in isolated nucleated peripheral blood cells (PBNCs) using real-time qPCR. Wet AMD was an independent factor associated with higher concentrations of IL-6 (β = +0.24, p = 0.0004), GM-CSF (β = +0.31, p < 0.001), IFN-γ (β = +0.58, p < 0.001), higher expression of miRNA-23a-3p (β = +0.60, p < 0.0001), miRNA-30b (β = +0.32, p < 0.0001), miRNA-191-5p (β = +0.28, p < 0.0001) and lower concentration of IL-1β (β = −0.25, p = 0.0003), IL-5 (β = −0.45, p < 0.001), IL-10 (β = −0.45, p < 0.001), IL-12 (β = −0.35, p < 0.001), lower expression of miRNA-16-5p (β = −0.31, p < 0.0001), miRNA-17-3p (β = −0.18, p = 0.01), miRNA-150-5p (β = −0.18, p = 0.01) and miRNA-155-5p (β = −0.47, p < 0.0001). Multivariate analysis revealed that dry AMD was an independent factor associated with higher concentration of GM-CSF (β = +0.34, p < 0.001), IL-6 (β = +0.13, p = 0.05), higher expression of miRNA-23a-3p (β = +0.60, p < 0.0001), miRNA-126-3p (β = +0.23, p = 0.0005), miRNA-126-5p (β = +0.16, p = 0.01), miRNA 146a (β = +0.14, p = 0.03), and mRNA191-5p (β = +0.15, p = 0.03) and lower concentrations of TNF-α (β = +0.24, p = 0.0004), IL-1β (β = −0.39, p < 0.001), IL-2 (β = −0.20, p = 0.003), IL-5 (β = −0.54, p < 0.001), IL-10 (β = −0.56, p < 0.001), IL-12 (β = −0.51, p < 0.001), lower expression of miRNA-16-5p (β = −0.23, p = 0.0004), miRNA-17-3p (β = −0.20, p = 0.003) and miRNA-17-5p (β = −0.19, p = 0.004). Negative correlations between visual acuity and WBC, lymphocyte count, TNF-α, IL-1 β, IL-2, IL-4, IL-6, IL-10 concentrations and miRNA-191-5p, as well as positive correlations between visual acuity and miRNA-126-3p, -126-5p, and -155-5p PBNCs expression were found in AMD patients. No such correlations were found in the control group. Our results may suggest the role of both intra- and extracellular mechanisms implicated in inflammatory response regulation in multifactorial AMD pathogenesis.

Platelet α-granules modulate the inflammatory response under systemic lipopolysaccharide injection in mice.

Beyond their role in hemostasis and thrombosis, platelets are also important mediators of inflammation by the release of hundreds of factors stored in their α-granules. Mutations in Nbeal2 cause gray platelet syndrome (GPS), characterized by the lack of platelet α-granules. This study aims to evaluate the immunological (proinflammatory) effects of platelet α-granules. We performed an experiment using Nbeal2-/- mice, the mouse model of GPS. Systemic inflammation was induced by intravenous injection of lipopolysaccharide (LPS). Inflammatory response was assessed by quantification of inflammatory soluble factors and platelet biological response modifiers. The lack of Nbeal2 (in Nbeal2 -/- mice, compared with controls) significantly reduced the recruitment of circulating neutrophils and monocytes. Moreover, after LPS injection, there was a significant increase in neutrophil and monocyte counts in control animals, compared with Nbeal2 -/- mice. The control of inflammation, evaluated by the production of anti-inflammatory cytokines, appeared to be greater in Nbeal2-/- mice compared with controls. Conversely, the production of certain inflammatory-soluble mediators known to characterize normal platelet secretion, such as soluble CD40 ligand (sCD40L), was decreased under experimental inflammation in Nbeal2 -/- mice. These results show that α-granules play a direct role in platelet-mediated inflammation balance, confirming the need to further investigate platelet-associated inflammatory pathophysiology and inflammatory adverse events related to blood transfusion.

1451 Development of a psoriatic immunocompetent organotypic 3D model as a new promising preclinical tool

The secretion of cytokine mediators between keratinocytes and T cells plays a major role in the development of chronic skin disease such as psoriasis. Some drugs that inhibit these inflammatory intermediates relatively improve the symptoms associated with psoriasis. To overcome the lack of a physiologically relevant organotypic model and to better understand psoriatic cellular and molecular interactions, we developed a unique reconstructed skin model in which positive CD3 T cells are seeded and interact with the three-dimensional local microenvironment. We analyzed the gene fluctuations by quantitative PCR and those of inflammatory proteins by Western Blot and Luminex of healthy, non-lesional and lesional reconstructed skin in which activated T lymphocytes were incorporated or not (controls). The epithelial phenotype of immunocompetent skin was altered, demonstrated by the decreased expression of keratin-10 and loricrin while involucrin, keratins 14 and 17 were upregulated. Protein overexpression of PCNA demonstrated the effect of T cells in inducing hyperproliferation of basal keratinocytes in tissue-engineered lesional skin. Finally, exacerbation of the expression of inflammatory soluble factors, such as IFN-γ, TNF-α, IL-17F, MCP-1 and that of transcription factors such as STAT1 and STAT3 underlined the role of lymphocytes in generating an inflammatory response. This first organotypic 3D model, validated by anti-inflammatory drug treatment, summarizing most of the key psoriatic characteristics, could therefore serve as a reliable preclinical tool for testing new therapeutic targets.

Modulation of the colon cancer cell phenotype by pro-inflammatory macrophages: A preclinical model of surgery-associated inflammation and tumor recurrence.

Peritoneal infection after colorectal cancer surgery is associated with a higher rate of tumor relapse. We have recently proposed that soluble inflammatory factors released in response to a postoperative infection enhance tumor progression features in residual tumor cells. In an effort to set up models to study the mechanisms of residual tumor cell activation during surgery-associated inflammation, we have analyzed the phenotypic response of colon cancer cell lines to the paracrine effects of THP-1 and U937 differentiated human macrophages, which release an inflammatory medium characteristic of an innate immune response. The exposure of the colon cancer cell lines HT-29 and SW620 to conditioned media isolated from differentiated THP-1 and U937 macrophages induced a mesenchymal-like phenotypic shift, involving the activation of in vitro invasiveness. The inflammatory media activated the β-catenin/TCF4 transcriptional pathway and induced the expression of several mesenchymal (e.g., FN1 and VIM) and TCF4 target genes (e.g., MMP7, PTGS2, MET, and CCD1). Similarly, differential expression of some transcription factors involved in epithelial-to-mesenchymal transitions (i.e. ZEB1, SNAI1, and SNAI2) was variably observed in the colon cancer cell lines when exposed to the inflammatory media. THP-1 and U937 macrophages, which displayed characteristics of M1 differentiation, overexpressed some cytokines previously shown to be induced in colorectal cancer patients with increased rates of tumor recurrence associated with postoperative peritoneal infections, thus suggesting their pro-tumoral character. Therefore, the environment created by inflammatory M1 macrophages enhances features of epithelial-to-mesenchymal transition, and may be useful as a model to characterize pro-inflammatory cytokines as putative biomarkers of tumor recurrence risk.

The CD38+ HLA-DR+ PD-1+ CD4 T cell population represents a short-lived part of the viral reservoir and predicts poor immunologic recovery upon initiation of ART

Abstract The activated PD-1+ CD4 T cell population is associated with disease progression in untreated HIV-1 infection, and with residual viral replication in individuals on ART. Few studies have examined cell associated viral load (CAVL) in phenotypically different activated CD4 T cell populations to define the HIV reservoir. We followed 32 HIV-1 chronically infected individuals, from Kampala, Uganda, and determined their CD4 T cell counts, and viral load at baseline, 6, and 12 months after the initiation of ART. Peripheral blood T cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble inflammatory factors were measured in plasma using a multiplexed platform. The median HIV-1 viral load declined from 95,315 copies/ml at baseline to below detection for all patients after 12 months of ART, whereas CD4 T cells counts averaged 204 cells/μl at baseline and increased to 373 cells/μl by month 12. Median levels of the activated PD-1+ CD4 T cell population contracted from 4.5% of CD4 T cells at baseline to 2.3% of CD4 T cells after 12 months of ART. Baseline levels of activated PD-1+ CD4 T cells positively correlated with plasma levels of IP-10 (R2=0.362, P&amp;lt;0.001) and TNFRII (R2=0.237, P=0.006). Higher baseline level of activated PD-1 positive CD4 T cells was associated with poorer CD4 T cell recovery after 12 months of ART (R2=0.240, P=0.039). CAVL was similar within the activated PD-1+ CD4 T cell population as other CD4 populations at baseline, but significantly lower after 12 months of ART compared to baseline. Taken together, the activated PD-1+ CD4 T cell population is a short-lived component of the viral reservoir and high levels of these cells predict poor immunologic recovery on ART.

Regular exercise during haemodialysis promotes an anti-inflammatory leucocyte profile.

BackgroundCardiovascular disease is the most common cause of mortality in haemodialysis (HD) patients and is highly predicted by markers of chronic inflammation. Regular exercise may have beneficial anti-inflammatory effects, but this is unclear in HD patients. This study assessed the effect of regular intradialytic exercise on soluble inflammatory factors and inflammatory leucocyte phenotypes.MethodsTwenty-two HD patients from a centre where intradialytic cycling was offered thrice weekly and 16 HD patients receiving usual care volunteered. Exercising patients aimed to cycle for 30 min at rating of perceived exertion of ‘somewhat hard’. Baseline characteristics were compared with 16 healthy age-matched individuals. Physical function, soluble inflammatory markers and leucocyte phenotypes were assessed again after 6 months of regular exercise.ResultsPatients were less active than their healthy counterparts and had significant elevations in measures of inflammation [interleukin-6 (IL-6), C-reactive protein (CRP), tumour necrosis factor-α (TNF-α), intermediate and non-classical monocytes; all P < 0.001]. Six months of regular intradialytic exercise improved physical function (sit-to-stand 60). After 6 months, the proportion of intermediate monocytes in the exercising patients reduced compared with non-exercisers (7.58 ± 1.68% to 6.38 ± 1.81% versus 6.86 ± 1.45% to 7.88 ± 1.66%; P < 0.01). Numbers (but not proportion) of regulatory T cells decreased in the non-exercising patients only (P < 0.05). Training had no significant effect on circulating IL-6, CRP or TNF-α concentrations.ConclusionsThese findings suggest that regular intradialytic exercise is associated with an anti-inflammatory effect at a circulating cellular level but not in circulating cytokines. This may be protective against the increased risk of cardiovascular disease and mortality that is associated with chronic inflammation and elevated numbers of intermediate monocytes.

Myeloid-derived suppressor cells and tumor escape from immune surveillance

Tumor progression is known to be supported by chronic inflammatory conditions developed in the tumor microenvironment. It is characterized by the long-term secretion of various inflammatory soluble factors (including cytokines, chemokines, growth factors, reactive oxygen and nitrogen species, prostaglandins, etc.) and strong leukocyte infiltration. Among leukocytes infiltrating tumors, myeloid-derived suppressor cells (MDSCs) represent one of the most important players mediating immunosuppression and supporting tumor escape. These cells can strongly inhibit antitumor immune reactions mediated by T cells and NK cells. Moreover, MDSCs are generated, recruited to the tumor site, and activated not only under the influence of soluble inflammatory mediators but also due to extracellular vesicles (EVs) secreted by tumor cells. EVs play a key role in the formation of MDSCs via the conversion of normal myeloid cells and altering the normal myelopoiesis. In addition, EVs help create a suitable microenvironment for the metastatic process.

PD-L1/PD-1 Immunotherapy Modulates Effector T Cells Homeostasis and Function in Murine Pancreatic Cancer

Abstract Pancreatic Cancer (PC) is one of the most aggressive and deadliest types of cancer, and it is projected to be the second leading cause of cancer-related deaths in the U.S. by the year 2030. PC evades immune surveillance by disrupting the immune homeostasis of effector T cells (Teff). T cells homeostasis is critical for proper anti-tumor immune responses. Preliminary flow cytometry data revealed that murine pancreatic (Panc02) cancer cells produce inflammatory soluble factors and in addition, express Major Histocompatibility Complex class I (MHC-I) and Programmed Death Ligand 1 (PD-L1) and 2 (PD-L2). PD-L1/2 can bind to PD-1 receptors on Teff and induce apoptosis or anergy, dampening the anti-tumor immunity. Cytokine bead array and flow cytometry analysis of serum from peripheral blood from C57BL/6 mice injected with Panc02 cells (TB mice) showed a significant increase in inflammatory factors compared to Control (CTRL) mice. Additionally, Teff from splenocytes from TB mice showed significant reduction in Teff percentages compared to CTRL mice, in vitro. In this study, CD3+ cells from CTRL mice were co-incubated with Panc02 cells pre-treated with anti-PD-L1 antibody (ab) to evaluate the modulation of Teff and cytokines production, in vitro. Mice were inoculated with the Panc02 pre-treated cells with anti-PD-L1 ab, to evaluate the modulation of Teff and cytokines production, in vivo. The results from this project may lead to the identification of new immunotherapeutic strategies to stabilize Teff homeostasis and function that could increase anti-tumor immune responses and combat PC progression.

Up-regulation of the monocyte chemotactic protein-3 in sera from bone marrow transplanted children with torquetenovirus infection

BackgroundTorquetenovirus (TTV) represents a commensal human virus producing life-long viremia in approximately 80% of healthy individuals of all ages. A potential pathogenic role for TTV has been suggested in immunocompromised patients with hepatitis of unknown etiology sustained by strong proinflammatory cytokines. ObjectivesThe aim of this study was to investigate the sera immunological profile linked to TTV infection in bone marrow transplant (BMT) children with liver injury. Study designTTV infection was assessed in sera from 27 BMT patients with altered hepatic parameters and histological features, by the use of quantitative real-time PCR, along with TTV genogroups and coinfection with HEV. The qualitative and quantitative nature of soluble inflammatory factors was evaluated studying a large set of cytokines using the Bioplex platform. As controls, sera from 22 healthy children negative for serological and molecular hepatitis markers including TTV and HEV, and for autoimmune diseases, were selected. Results and conclusionsTTV was detected in 81.4% of BMT patients with a viral load ranging from 105 to 109 copies/mL. All samples were HEV-RNA negative. A pattern of cytokines, IFN-γ, TNF-α, FGF-basic (p<0.01) and MCP-3 (p<0.001) was found significantly highly expressed in TTV-positive patients compared to TTV-negative and controls. Of note, MCP-3 chemokine showed the highest sera concentration independently of the amount of TTV load and the status of immune system deregulation (p<0.001). In this pilot study for the first time, a positive association was found between TTV and increased level of MCP-3 suggesting a indirect role of TTV in liver injury.