CCL2 Levels Research Articles

CCL19+ dendritic cells potentiate clinical benefit of anti-PD-(L)1 immunotherapy in triple-negative breast cancer

The extensive involvement of dendritic cells (DCs) in immune contexture indicates their potent value in cancer immunotherapy. Understanding DC diversity in patient cohorts may strengthen the clinical benefit of immune checkpoint inhibitors (ICIs). Single-cell profiling of breast tumors from two clinical trials was performed to investigate DC heterogeneity. Multiomics, tissue characterization, and pre-clinical experiments were used to evaluate the role of the identified DCs in the tumor microenvironment. Four independent clinical trials were leveraged to explore biomarkers to predict ICI and chemotherapy outcomes. We identified a distinct CCL19-expressing functional state of DCs associated with favorable responses to anti-programmed death (ligand)-1 (PD-(L)1), which displayed migratory and immunomodulatory phenotypes. These cells were correlated with antitumor Tcell immunity and the presence of tertiary lymphoid structures and lymphoid aggregates, defining immunogenic microenvironments in triple-negative breast cancer. Invivo, CCL19+ DC deletion by Ccl19 gene ablation dampened CCR7+CD8+ Tcells and tumor elimination in response to anti-PD-1. Notably, high circulating and intratumoral CCL19 levels were associated with superior response and survival in patients receiving anti-PD-1 but not chemotherapy. We uncovered a critical role of DC subsets in immunotherapy, which has implications for designing novel therapies and patient stratification strategies. This study was funded by the National Key Research and Development Project of China, the National Natural Science Foundation of China, the Program of Shanghai Academic/Technology Research Leader, the Natural Science Foundation of Shanghai, the Shanghai Key Laboratory of Breast Cancer, the Shanghai Hospital Development Center (SHDC), and the Shanghai Health Commission.

High-fat diet promotes multiple binges-induced liver injury via promoting hepatic macrophage proinflammatory polarization.

High-fat diet (HFD) and ethanol could synergistically induce liver damage, but the underlying mechanisms remain to be elucidated. M1-polarized macrophages have been demonstrated to be key players in ethanol-induced liver damage. The current study was designed to investigate whether hepatic steatosis could promote ethanol-induced liver injury by promoting liver macrophage M1 polarization. In the in vivo study, 12weeks of HFD feeding induced a moderate increase in the F4/80 expression and protein levels of p-IKKα/β, p-IκBα, and p-p65, which was suppressed by single binge. In contrast, 8weeks of HFD and multiple binges (two binges per week during the last 4weeks) synergistically increased the F4/80 expression, mRNA levels of M1 polarization biomarkers including Ccl2, Tnfa, and Il1b, and protein levels of p65, p-p65, COX2, and Caspase 1. In the in vitro study, a nontoxic free fatty acids (FFAs) mixture (oleic acid/palmitic acid=2: 1) induced a moderate increase of protein levels of p-p65 and NLRP3 in murine AML12 hepatocytes, which was inhibited by ethanol co-exposure. Ethanol alone induced proinflammatory polarization of murine J774A.1 macrophages evidenced by the enhanced secretion of TNF-α, increased mRNA levels of Ccl2, Tnfa, and Il1b, and upregulated protein levels of p65, p-p65, NLRP3, and Caspase 1, which was augmented by FFAs exposure. Collectively, these results suggest that HFD and multiple binges could synergistically induce liver damage by promoting the proinflammatory activation of macrophages in mice livers.

CCL18 as a Biomarker of Interstitial Lung Disease (ILD) and Progressive Fibrosing ILD in Patients with Idiopathic Inflammatory Myopathies.

To assess CCL18 and OX40L as biomarkers of interstitial lung disease (ILD) and/or progressive fibrosing (PF-) ILD in idiopathic inflammatory myopathies (IIMs). Patients with IIMs seen in our center from July 2020 to March 2021 were consecutively enrolled. ILD was detected by high-resolution CT. CCL18 and OX40L serum levels were measured by validated ELISA assays in 93 patients and 35 controls. At the 2-year follow-up, PF-ILD was evaluated according to the INBUILD criteria. ILD was diagnosed in 50 (53.7%) patients. CCL18 serum levels were higher in IIMs patients vs. controls (232.9 [IQR 134.7-399.07] vs. 48.4 [29.9-147.5], p < 0.0001), with no difference for OX40L. IIMs-ILD patients exhibited higher levels of CCL18 than those without ILD (306.8 [190.8-520.5] vs. 162 [75.4-255.8], p < 0.0001). High CCL18 serum levels were independently associated with IIMs-ILD diagnosis. At follow-up, 22/50 (44%) patients developed a PF-ILD. Patients who developed PF-ILD had higher CCL18 serum levels than non-progressors (511 [307-958.7] vs. 207.1 [149.3-381.7], p < 0.0001). Multivariate logistic regression analysis revealed CCL18 as the only independent predictor of PF-ILD (OR 1.006 [1.002-1.011], p = 0.005). Although in a relatively small sample, our data suggest that CCL18 is a useful biomarker in IIMs-ILD, particularly in the early identification of patients at risk of developing PF-ILD.

Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

CCL2 promotes lymphatic metastasis via activating RhoA and Rac1 pathway and predict prognosis to some extent in tongue cancer

ABSTRACT Background Oral tongue cancer is an extremely malignant tumor with high rate of lymphatic metastasis. Little is known about its invasion and metastasis mechanisms so far. Methods To clarify the main role of CCL2 in tongue cancer progression, we performed Transwell migration assay to confirm the effects of different concentrations of CCL2 on the migration and invasion of tongue cancer cells. Next, by siRNA interference of RhoA and Rac1 in LNMTca8113 cells, we are able to observe that these two molecules block the effect of CCL2 on cell migration and cytoskeleton reorganization by laser confocal microscopy. Moreover, the AKT phosphorylation level of PI3K downstream molecule under the action of CCL2 also be detected by qRT-PCR and western blot, so as to determine whether CCL2 affects the proliferation of LNMTca8113 cells through PI3K/AKT pathway. Finally, we analyzed the relationship between plasma CCL2 level and various clinicopathological parameters in patients with tongue cancer. Results: We found that tongue cancer cells treated with CCL2 migrate faster at first. CCL2 may enhance the invasion and migration of LNMTca8113 cells by activating RhoA and Rac1 to promote cytoskeleton reorganization. Promotion of LNMTca8113 migration induced by CCL2 was inhibited by silence of RhoA and Rac1. CCL2 could increase the phosphorylation of downstream Akt/PI3K signal and promoted the proliferation. Plasma concentration confirmed that the CCL2 level was closely related to the clinical stage of tongue cancer. Patients with lower CCL2 levels had a relatively longer progression-free survival and total survival time. Results After adding CCL2, the number of proliferation and migration of tongue cancer cells increased and the expression of RhoA and Rac1 rose up in LNMTca8113 cell line. The cytoskeleton reorganization was notable. Patients with higher serum levels of CCL2 had shorter progression-free survival than those with lower levels of CCL2 (P < 0.0001). Conclusions CCL2 promotes the invasion and metastasis of tongue cancer by PI3K/Akt pathway. The plasma level of CCL2 may predict prognosis of tongue cancer patients. CCL2 can serve as a potential therapeutic target for tongue cancer treatment.

The role of inflammation in anxiety and depression in the European U-BIOPRED asthma cohorts

BackgroundGrowing evidence indicates high comorbid anxiety and depression in patients with asthma. However, the mechanisms underlying this comorbid condition remain unclear. The aim of this study was to investigate the role of inflammation in comorbid anxiety and depression in three asthma patient cohorts of the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) project. MethodsU-BIOPRED was conducted by a European Union consortium of 16 academic institutions in 11 European countries. A subset dataset from subjects with valid anxiety and depression measures and a large blood biomarker dataset were analysed, including 198 non-smoking patients with severe asthma (SAn), 65 smoking patients with severe asthma (SAs), 61 non-smoking patients with mild-to-moderate asthma (MMA), and 20 healthy non-smokers (HC). The Hospital Anxiety and Depression Scale was used to measure anxiety and depression and a series of inflammatory markers were analysed by the SomaScan v3 platform (SomaLogic, Boulder, Colo). ANOVA and the Kruskal-Wallis test were used for multiple-group comparisons as appropriate. ResultsThere were significant group effects on anxiety and depression among the four cohort groups (p < 0.05). Anxiety and depression of SAn and SAs groups were significantly higher than that of MMA and HC groups (p < 0.05. There were significant differences in serum IL6, MCP1, CCL18, CCL17, IL8, and Eotaxin among the four groups (p < 0.05). Depression was significantly associated with IL6, MCP1, CCL18 level, and CCL17; whereas anxiety was associated with CCL17 only (p < 0.05). ConclusionsThe current study suggests that severe asthma patients are associated with higher levels of anxiety and depression, and inflammatory responses may underlie this comorbid condition.

Differentiated Embryonic Neurospheres from Familial Alzheimer's Disease Model Show Innate Immune and Glial Cell Responses.

Proteins involved in the Alzheimer's disease (AD), such as amyloid precursor protein (APP) and presenilin-1 (PS1), play critical roles in early development of the central nervous system (CNS), as well as in innate immune and glial cell responses. Familial AD is associated with the presence of APPswe and PS1dE9 mutations. However, it is still unknown whether these mutations cause deficits in CNS development of carriers. We studied genome-wide gene expression profiles of differentiated neural progenitor cells (NPCs) from wild-type and APPswe/PS1dE9 mouse embryo telencephalon. The occurrence of strong innate immune and glial cell responses in APPswe/PS1dE9 neurospheres mainly involves microglial activation, inflammatory mediators and chemokines. APPswe/PS1dE9 neurospheres augmented up to 100-fold CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF-alpha expression levels, when compared to WT neurospheres. Expression levels of the glia cell marker GFAP and microglia marker Iba-1 were up to 20-fold upregulated in APPswe/PS1dE9 neurospheres. The secretome of differentiated APPswe/PS1dE9 NPCs revealed enhanced chemoattraction of peripheral blood mononuclear cells. When evaluating the inferred protein interaction networks constructed from the array data, an improvement in astrocyte differentiation in APPswe/PS1dE9 neurosphereswas evident in view ofincreased GFAPexpression. Transgenic NPCs differentiated into neural phenotypes presented expression patterns of cytokine, glial cells, and inflammatory mediators characteristic of APPswe/PS1dE9 adult animals. Consequently, the neurogenic niche obtained from differentiation of embryonic APPswe/PS1dE9 neurospheres spontaneously presents several alterations observed in adult ADbrains. Finally, our data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for familial AD.

Abstract 284: Pro-Inflammatory Adventitial Fibroblast Contribute To Aortic Aneurysms In Loeys Dietz Syndrome

Introduction: Vascular smooth muscle cell (SMC) phenotype modulation is implicated in thoracic aneurysm formation, however, the role of adventitial fibroblasts (AdvFib) in aneurysm formation remains poorly characterized. Loeys-Dietz Syndrome (LDS), caused by a spectrum of mutations in the TGF-β signaling cascade, leads to aggressive thoracic aneurysms. Given the ubiquitous nature of TGF-β signaling machinery, the exact cell type(s) responsible for initiation of LDS pathogenesis is poorly understood. Methods: We performed single cell transcriptomic characterization of aortic root and ascending aortic samples from LDS patients ((n=6 LDS (harboring TGFB2 , TGFBR1 , TGFBR2 , or SMAD3 mutation); n=2 Donor control)) as well as root/ascending aorta from a murine model of LDS ( Tgfbr2 G357W/+ ) to understand cell-state transitions and transcriptomic alterations in LDS. Results: Single cell RNA sequencing (scRNAseq) of human and mice LDS samples (in total &gt;40,000 cells) revealed a secondary pattern of cellular activation in LDS as compared to Marfan Syndrome. Instead of SMC phenotypic modulation as seen in MFS, LDS appears to preferentially modulate adventitial fibroblasts (AdvFib) phenotype in the Tgfbr2 G357W/+ mouse model. Human LDS patient’s AdvFib turn on high levels of macrophage-recruiting pro-inflammatory cytokines CCL2 and HLA-B, with a corresponding 1.8-fold increase in LDS aortic wall macrophages compared to Donor. A more extreme pattern was observed in the Tgfbr2 G357W/+ LDS mouse model. While Tgfbr2 G357W/+ SMC are transcriptomically similar to wild type counterparts, AdvFib were activated into a pro-inflammatory state associated with increased aortic macrophage recruitment ( Ccl2 , Il6 , Vcam1, and Cxcl2 ) and fibrotic response ( Col1a1 , Col1a2 , Col3a1, and Fn1 ). A 6-fold increase in the number of aortic wall macrophages was identified in Tgfbr2 G357W/+ compared to WT. Conclusions: LDS mouse and human AdvFib undergo phenotypic alterations into a pro-inflammatory state evidenced by increased expression of numerous cytokines, macrophage recruitment, and ECM deposition. AdvFib, in addition to SMCs, are potentially another important pathological cell population in LDS aneurysm formation.

Novel Biomarker Evaluation in Peri-Implant Crevicular Fluid as Disease Indicators for Peri-Implant Health

Objective: To assess various inflammatory biomarkers CCL-20, BAF, RANK-L, IL-23, and osteoprotegerin from PICF along with clinical parameters in participants with peri-implant mucositis and peri-implantitis. Material and Methods: Participants were gathered i.e., 30 with periimplantitis, 32 with peri-implant mucositis, and 32 healthy. Peri-implant parameters PIPD, BoP, and PIPI were measured in all participants. PICF samples were collected to assess the level of biomarkers CCL-20; BAF; IL-23; RANK-L, and OPG. For periodontal parameters, ranges and means were measured. Kruskal Wallis test was used for comparison between groups. For categorical data sets, the Pearson Chi-Square test was applied. The Bonferroni test was used for multiple comparisons. Results: Peri-implant parameter BoP in peri-implant mucositis and peri-implantitis was significantly higher compared to controls (p &lt; 0.05). PIPD with healthy peri-implant conditions was, significantly less compared to peri-implantitis, and peri-implant mucositis (p&lt;0.05). PIPI demonstrated no significant difference throughout different peri-implant conditions. CCL-20 ng/mL in patients with peri-implant mucositis was found to be significantly higher (p &lt; 0.05). BAF levels in peri-implantitis and peri-implant mucositis were comparable. The concentration of IL-23 ng/mL was found to be significantly lower in healthy controls (p &lt; 0.05). Conclusion: Inflammatory biomarkers showed high levels of PICF in peri-implant disease patients compared to healthy controls. Individuals with peri-implant conditions experience the poor peri-implant probing depth and bleeding on probing.

82 Responses to a combined exercise and transport stressor following dietary supplementation of anti-endotoxin IgY in mature horses

Immunoglobulin Y elicits positive effects in domestic livestock with gastrointestinal or inflammatory diseases; however, there are limited studies in horses. To test the hypothesis that dietary anti-endotoxin IgY (IgY) has a positive effect on gastrointestinal health and mitigates systemic inflammation in horses undergoing exercise, 30 stock-type horses (578 ± 59 kg BW; 14 ± 3 yr) were used in a completely randomized design. Horses were stratified by BW, age, and sex and then randomly assigned to treatments: 0 g/d IgY (CON; n = 10), 1 g/d IgY (TRT1; n = 10), or 2 g/d IgY (TRT2; n = 10). Horses were fed every 12 h, with dietary treatments (Camas Inc., Le Center, MN) top-dressed on the morning concentrate for 33 d. Horses were maintained in dry lots with ad libitum access to Coastal bermudagrass hay. Horses were exercised 5 d/wk for 1 h. On d 0 and 30, BW and BCS were recorded, and blood was collected for complete blood cell count (CBC) and serum blood chemistry. On d 32, horses were transported 270 km and underwent a 3.2 km exercise stressor on concrete at a walk with maximum 15 min of trot. Whole blood, serum, and fecal samples collected via rectal palpation were obtained pre-, post-, and 24 h post-trailering. Fecal pH was determined by a portable meter, and serum cytokine (TNFα, IL-4, IL-10, IL-1β, IL-17, IFN-α, IFN-γ) and chemokine (CCL2, 3, 5 and 11) concentrations weremeasured using a multiplex platform. Whole blood DNA was extracted using a commercial DNA extraction kit. For gut permeability analysis, 16s rDNA was analyzed via qPCR and calculated as fold change from d 0. Data were analyzed using PROC MIXED of SAS v9.4. Dietary IgY did not affect BW, BCS, blood chemistry, or CBC (P ≥ 0.17). Fecal pH decreased over time (P ≤ 0.01). There was no treatment × time interaction for 16S rDNA (P = 0.45). Dietary treatment affected circulating 16S rDNA (P < 0.01) where TRT2 (1.89-fold) was greater than CON (0.70-fold), and TRT1 (1.24-fold) was intermediate. A significant time effect (P = 0.001) was observed for 16S rDNA where fold change decreased after the stressor from 1.90-fold for pre-, to 1.02 for post-, and 0.92 for 24 h post- trailering. There tended to be a treatment × time interaction (P = 0.08) for CCL3, as CON was greater at 24 h and TRT2 declined post-trailering, returning to baseline by 24 h. Levels of CCL5 decreased (P < 0.01) and TNFα tended to decline (P = 0.06) through 24 h post trailering in all treatment groups. Other cytokines and chemokines were non-detectible. Despite greater circulating 16s rDNA with higher treatment dosages, there was no effect on systemic inflammatory markers.

TLR-mediated immune activation is a strong suppressor of the Treg-attracting chemokine CCL22

Abstract The immunoregulatory chemokine CCL22 is mainly secreted by dendritic cells (DC) in lymph nodes and plays a crucial role in maintaining the balance between immune tolerance and efficient immune response by induction of intercellular contacts between DC and regulatory T cells (Treg) expressing the only so far known CCL22 receptor CCR4. In the absence of CCL22, the immune response to vaccination and cancer has been proven to be enhanced. In this work, we aimed to understand how activation of Toll-like receptors (TLR) modulates CCL22 levels mechanistically in vitro and in vivo. We observed that a bacterial infection with salmonella typhimurium and treatment with different TLR agonists such as Poly (I:C), LPS, R848 and CpG led to a potent and long-lasting suppression of systemic CCL22 in vivo. Furthermore, using in vitro co-cultures of DC, B cells and T cells, we identified TLR-mediated CCL22 suppression to be mediated by soluble factors derived from non-DC. Additionally, we pursued to investigate the underlying mechanism and could show in vitro that CCL22 suppression is particularly driven by redundant action of IL-10, IFN-α and IFN-γ. Moreover, inhibition of NF-κB reverted the CCL22-suppressing effect of CpG on splenocytes. In CpG-treated mice, Treg/DC contacts were diminished to the level known from Ccl22 −/−mice, an observation which links TLR-mediated CCL22 suppression to an immunological relevance. Taken together, we demonstrate that TLR signaling results in CCL22 downregulation via different cytokines, a mechanism which is potentially important to enable the induction of a strong immune response and efficient pathogen clearance in the setting of microbial infection.

Commensal probiotic provides long-term protection in a murine model of allergic lung disease by altering the host microbiota

Abstract The incidence of allergic diseases is increasing, with house dust mite allergy, afflicting up to 20% of the population in developed countries. House dust mites (HDM) are a common allergen that can be difficult to avoid and triggers allergic reactions, including wheezing and shortness of breath. Bacillus subtilis is a gut-associated commensal probiotic that can prevent murine HDM-induced allergic lung inflammation. We exposed C57Bl/6 mice to B. subtilis via oral gavage and challenged with intranasal HDM for up to 8 weeks. We found that pretreatment with B. subtilis was able to provide protection from eosinophil infiltration, measured in bronchial alveolar lavage fluid (BALF), for 3 weeks. This loss of protection correlated with an increase in the eosinophil chemoattractant CCL24 levels in the BALF and reduced abundance of the bacterial phylum Verrucomicorbiota in collected feces. Additionally, we found that B. subtilis treatment altered the bacterial composition by increasing the abundance of the bacteria phylum Bacteroidetes. These results support B. subtilis as a prophylactic for preventing allergic lung disease and highlights that protection can last up to 3 weeks. This work also expands our understanding of how B. subtilis mediates protection and that in addition to modifying the immune system, it is also altering the host microbiota. This work is supported by start-up funds and core facility outsourcing funds from Midwestern University.

Interstitial lung disease and mycobacteriosis in children with inherited CCR2 deficiency

Tissue-resident alveolar macrophages are important for lung homeostasis and innate immunity. They originate from fetal monocytes and are maintained in the lung through self-renewal or may arise from recruited circulating monocytes. Disturbance of alveolar macrophage function, their numbers or loss of GM-CSF signaling can all underlie pulmonary alveolar proteinosis (PAP). Here, we report eight patients (aged 8–21 years) from four kindreds and three ancestries suffering from recurrent pneumonia, progressive interstitial fibrosis, and PAP. All patients are deficient for the monocyte-chemoattractant protein-1 (CCL-2) receptor 2 (CCR2). Five patients are homozygous and three are compound heterozygous for private or rare CCR2 variants. The variants are loss-of-function for Ca2 +-release and cell migration upon CCL-2 stimulation, when tested by overexpression in monocytic THP-1 cells. Moreover, CCL-2-dependent migration and Ca2+-release of the patients’ monocytes are abolished, while their plasma CCL-2 levels are elevated. Yet, CCR2-deficient cells respond normally to GM-CSF. In addition, the counts, frequencies, and transcriptomes of blood lymphoid and myeloid subsets are normal. Mice deficient for CCR2 display alveolar septal wall thickening and increased number of alveolar type II cells producing surfactant protein C causing increased lung tissue hysteresis. CCR2-deficient mice are vulnerable to mycobacteria, with impaired attraction of blood monocytes to infected tissues. These findings suggest that human CCR2 is essential for the recruitment of monocytes to both pulmonary alveoli and tissues infected with mycobacteria.

Synergism between chikungunya virus infection and rheumatoid arthritis on cytokine levels: Clinical implications?

Post-chikungunya virus (CHIKV) chronic arthritis shares several immunopathogenic mechanisms with rheumatoid arthritis (RA), which has led to discussions about the probable relationship between the two diseases. Indeed, some studies have suggested a role for CHIKV infection in RA development. However, to the best of our knowledge, the influence of CHIKV on previous RA has not yet been demonstrated. Herein, we analyzed the potential synergism between CHIKV infection and RA on cytokine and chemokine levels. For this, we compared the IL-1β, IL-6, IL-10, IL-17A, CCL2, CXCL8, CXCL9 and CXCL10 levels, in addition to rheumatoid factor (RF) and C-reactive protein (CRP), in patients with post-CHIKV chronic arthritis (named CHIKV group), patients with RA (RA group), and patients with previous RA who were later infected by CHIKV (RA-CHIKV). History of CHIKV infection was confirmed by serology (IgG, ELISA). Cytokines/chemokines were quantified by flow cytometry. RF, CRP, age and sex data were obtained from medical records. IL-1β, IL-6, IL-10 and IL-17A levels were significantly higher in RA-CHIKV compared to the other groups. CXCL8 levels were higher in the CHIKV group than in RA. CXCL9 was higher in CHIKV than in the RA-CHIKV group. CXCL10 was higher in CHIKV than in the other groups. FR levels were higher in RA than in the CHIKV group, and in RA-CHIKV than in CHIKV. No significant difference was observed in CCL2 and CRP, as well as in age and sex. Finally, our findings suggest an interplay between CHIKV infection and RA, which must be analyzed for its possible clinical impact.

Immunomodulatory effects of trans-anethole-treated Staphylococcus aureus Newman strain

In our former studies based on a human whole-blood model infected with trans-anethole (TA)-treated Staphylococcus aureus Newman strain, we have observed that selected parameters/mechanisms of innate and acquired immune response were more enhanced in comparison to samples infected with non-treated bacteria. Due to this observation, the current study aimed to evaluate the concentration of selected proteins involved in both types of responses (IL-1α, IL-1β, IL-2, IL-6, IL-12, IL-17, TNF-α, IFN-γ, G-CSF, C5a, CCL1—CCL5, CXCL1, CXCL2, CXCL9—CXCL11, MMP-8, TLR2, and PGLYRP1) in healthy participants' plasma after blood stimulation of TA-treated S. aureus Newman strain. Determination of analyzed protein concentration was conducted using Luminex and ELISA assays. Based on the results, it has been proven that the immunomodulatory potential of TA-treated S. aureus Newman strain on increasing IL-1β, IL-6, TNF-α, IL-12, G-CSF, C5a, CCL2—CCL4, CXCL1, CXCL2, MMP-8 and PGLYRP1 levels in plasma. Moreover, it has been also demonstrated an association between TNF-α and CCL4 in a blood model infected with TA-treated cells. More research is warranted to find more underlying mechanisms involved in the effects of TA-treated S. aureus Newman in human blood, mainly whether the observed “immunity boost” can be regulated after bacteria elimination. Therefore, the potential of TA should be further explored to understand under which conditions it might help treat or prevent infections caused by S. aureus.

OA28 CCL24 serum concentration predicts both vascular and fibrotic complications in systemic sclerosis: rationale for a biological target driven basket trial across disease subsets

Abstract Background/Aims CCL24 is a novel target that was found to play a dual role in advancing pro-inflammatory and pro-fibrotic processes in systemic sclerosis (SSc). Previous studies reported that skin and serum CCL24 levels are elevated in SSc and that blocking of CCL24 was effective in preventing and attenuating experimental-induced fibrosis in murine models as well as interfering with endothelial cell activation. Here we tested the concentration of CCL24 in an observational cohort of patients with SSc and available clinical follow-up to determine its clinical value in predicting fibrotic and vascular complications of the disease. Methods Consecutive patients from a single-centre observational cohort were enrolled in this analysis. Clinical data were collected according to the EUSTAR minimal essential data set. Serum Concentration of CCL24 was measured by Luminex assay (Myriad RBM) and defined as “high” for patients over the upper quartile of the healthy control population (110 ng/ml). Interstitial lung disease (ILD) was defined by at least 10% parenchymal involvement at a high-resolution CT scan of the chest, while digital ulcer (DU) disease was defined by the presence of current or history of DU in the previous 6 months. Progressive fibrotic ILD was defined as previously described, according to Erice's criteria within 24 months. Clinical features were compared between CCL24 high vs low by Chi-Square and T-student tests. R software was employed for statistical analysis. Results 189 SSc patients (13% male) fulfilling 2013 ACR/EULAR classification criteria were enrolled in this study. Median (IQR) disease duration was 4 (IQR 2-10) years. 37 (27%) had diffuse cutaneous SSc, 60 (32%) had ILD, and 90 (48%) had DU disease. 52 patients (26%) were CCL24 High. These patients had a higher prevalence of DU (60%, p = 0.042), a higher median (IQR) mRSS [4(2-7) vs 2(0-5), p = 0.036) and showed a higher prevalence of ILD (42% vs 28%, p = 0.055); this was more significant among patients with limited cutaneous SSc (7% vs 12%, p = 0.037). Most importantly, among patients with PF-ILD (19/43 with available 24-month follow-up), 44% had significantly higher CCL24 serum concentration at baseline, compared to stable patients (1150 [817-1593] vs 725 [460-955]; p = 0.017). Consistent with these findings, in patients with ILD, having high CCL24 gave double the chance of progressing within 24 months (66% vs 32% of CCL24 low; p = 0.03). Conclusion We show here that within the context of an observational cohort of unselected patients, CCL24 serum concentration is associated with the presence of DU as well as worse skin and lung fibrotic involvement, suggesting that this molecule may be involved in both vascular and fibrotic manifestations of SSc. These results informed the rationale for a target-enriched basket randomized controlled trial to test the biological and clinical effect of CCL24 inhibition in SSc across cutaneous disease subsets. Disclosure E. De Lorenzis: None. A. Mor: None. R. Ross: None. S. Di Donato: None. R. Aricha: None. H. Levi: None. I. Vaknin: None. F. Del Galdo: Other; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

Upregulation of IL-33, CCL2, and CXCL16 levels in Brazilian pregnant women infected by Toxoplasma gondii

Congenital toxoplasmosis can cause neurological and eye damage, behavioral alterations, or death in fetuses or babies born to Toxoplasma gondii-infected women. Several pieces of evidence suggest that socioeconomic, environmental, and inflammatory patterns linked to the maternal immune response partly drive the pathogenesis of this disease. However, immunoregulation induced by T. gondii infection during gestation is not completely understood. The aim of this study was to assess the association between T. gondii seropositivity and concentrations of plasma markers (CCL2, CXCL16, IL-17, and IL-33) in Brazilian pregnant women. Inflammatory markers were measured by immunoassays in the plasma of 131 pregnant women (13 to 46 years old). The prevalence of T. gondii infections was 45.8% (n = 60) in this population. The concentrations of CCL2, CXCL16, and IL-33 were higher in T. gondii-seropositive than in seronegative pregnant women, while the opposite was observed for IL-17 levels. In IgG+ women, a strong correlation between IL-17 and IL-33 (r = 0.7508, p = 0.0001) and a moderate correlation between CXCL16/IL-17 (r = 0.7319, p = 0.0001) and CXCL16/CCL2 (r = 0.3519, p = 0.0098) was observed. In uninfected women, a strong correlation was found between IL-17 and CXCL16 (r = 0.6779, p = 0.0001) but moderate between IL-17 and IL-33 (r = 0.4820, p = 0.0001). In summary, our data suggest that plasma upregulation of CCL2, CXCL16, and IL-33 might exert a potential protective role in the mother/fetus/parasite axis and, in addition, multiparous women are more likely to be infected with T. gondii than primiparous women.

Fulvic Acid Attenuates Atopic Dermatitis by Downregulating CCL17/22.

The main pathogenic factor in atopic dermatitis (AD) is Th2 inflammation, and levels of serum CCL17 and CCL22 are related to severity in AD patients. Fulvic acid (FA) is a kind of natural humic acid with anti-inflammatory, antibacterial, and immunomodulatory effects. Our experiments demonstrated the therapeutic effect of FA on AD mice and revealed some potential mechanisms. FA was shown to reduce TARC/CCL17 and MDC/CCL22 expression in HaCaT cells stimulated by TNF-α and IFN-γ. The inhibitors showed that FA inhibits CCL17 and CCL22 production by deactivating the p38 MAPK and JNK pathways. After 2,4-dinitrochlorobenzene (DNCB) induction in mice with atopic dermatitis, FA effectively reduced the symptoms and serum levels of CCL17 and CCL22. In conclusion, topical FA attenuated AD via downregulation of CCL17 and CCL22, via inhibition of P38 MAPK and JNK phosphorylation, and FA is a potential therapeutic agent for AD.

Distinct circulating cytokine/chemokine profiles correlate with clinical benefit of immune checkpoint inhibitor monotherapy and combination therapy in advanced non-small cell lung cancer.

An ever-increasing number of efforts are focused on identifying effective biomarkers for immune checkpoint inhibitors (ICIs). Cytokines and chemokines are critical to tumor growth, metastasis, tumor angiogenesis, and the immune response against tumor cells. In the study here, we determined the correlation between circulating cytokines/chemokines and the clinical benefit of ICIs for non-small cell lung cancer (NSCLC) patients. Peripheral blood samples were collected before and during treatment (at 12th week). Plasma levels of cytokines/chemokines and specific stress response markers were measured using the Bio-Plex Pro Human Cytokines Grp I Panel (27-plex), an APEX1 detection kit, and a human LAP(TGF-β1) immunoassay kit. A Mann-Whitney U-test or Wilcoxon signed-rank test and a Cox proportional hazards model were employed for statistical analysis. In the ICI monotherapy cohort, a high level of IL-6 at pretreatment or an elevation of IL-6, IL-8, FGF2, CXCL10, CCR1, PDFGB, TNF, and APEX1 posttreatment was associated with poor progress-free survival (PFS). A posttreatment elevation (defined herein as change rate) of CXCL10 was also associated with poor overall survival (OS). In the combinational therapy group, a high level of IL-12, IL-17A, FGF2, VEGF, and APEX1 at pretreatment and an elevation of CCL2 posttreatment were associated with poor PFS. A high level of IL-9, FGF2, PDFGB, CCL4, TFGB, and APEX1 at pretreatment and an elevation of IL-13, CSF2, and CCL2 at posttreatment were associated with poor OS of patients receiving combination therapy. The study here suggests that circulating cytokines/chemokines are feasible, noninvasive biomarkers for predicting clinical benefit of ICI treatment for NSCLC. Distinct circulating factor profiles were observed in individuals receiving ICI monotherapy or combination therapy.

Identification and validation of chemokine system-related genes in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease with limited therapeutic options. Recent studies have demonstrated that chemokines play a vital role in IPF pathogenesis. In the present study, we explored whether the gene signature associated with chemokines could be used as a reliable biological marker for patients with IPF. Chemokine-related differentially expressed genes (CR-DEGs) in IPF and control lung tissue samples were identified using data from the Gene Expression Omnibus database. A chemokine-related signature of the diagnostic model was established using the LASSO-Cox regression. In addition, unsupervised cluster analysis was conducted using consensus-clustering algorithms. The CIBERSORT algorithm was used to calculate immune cell infiltration across patient subgroups. Finally, we established a mouse model of bleomycin-induced pulmonary fibrosis and a model of fibroblasts treated with TGFβ1. Expression levels of chemokine-related signature genes were determined using real-time quantitative polymerase chain reaction (RT-qPCR). We established a chemokine-related eleven-gene signature of a diagnostic model consisting of CXCL2, CCRL2, ARRB1, XCL1, GRK5, PPBP, CCL19, CCL13, CCL11, CXCL6, and CXCL13, which could easily distinguish between IPF patients and controls. Additionally, we identified two subtypes of IPF samples based on chemokine-related gene expression. Pulmonary function parameters and stromal scores were significantly higher in subtype 1 than in subtype 2. Several immune cell types, especially plasma cells and macrophages, differ significantly between the two subtypes. RT-qPCR results showed that the expression levels of Cxcl2 and Ccl2 increased considerably in bleomycin-induced mice. Meanwhile, Arrb1, Ccrl2, Grk5, and Ppbp expression was significantly reduced. Furthermore, multiple chemokine-related genes were altered in TGFβ1 or TNFα-induced fibroblast cells. A novel chemokine-related eleven-signature of diagnostic model was developed. These genes are potential biomarkers of IPF and may play essential roles in its pathogenesis.