Sphingosine-1-phosphate (S1P) and its receptors have been implicated in functions of Langerhans cells and atopic dermatitis. In this study, we investigated the roles of S1P receptor type 2 (S1P₂) in a mouse model of atopic dermatitis, which was induced by topical application of 2,4-dinitrochlorobenzene (DNCB) on ventral skin on D0, followed by repeated DNCB challenge on both ears from D7 to D49. Wild-type mice with atopic dermatitis displayed severe inflammation and mast cell accumulation in ear tissues and elevated IgE levels in serum. Furthermore, the mice showed significantly increased sizes of draining lymph nodes, high levels of inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in the ears and lymph nodes and high levels of chemokines CCL17 and CCL22 in ears. Administration of JTE-013, a selective antagonist of S1P₂ (3 mg/kg, i.p, from D19 to D49) before DNCB challenge significantly suppressed DNCB-induced atopic responses in ears and lymph nodes. JTE-013 administration also significantly decreased the lymph nodes sizes, the levels of inflammatory cytokines (IL-4, IL-13, IL-17, and IFN-γ) in the ears and lymph nodes, and the levels of chemokines CCL17 and CCL22 in ears. Furthermore, the inflammatory responses of atopic dermatitis were greatly ameliorated in S1pr2 gene-deficient mice. As CCL17 and CCL22 are CCR4 ligands, acting as Th2-attracting chemokines, we investigated CCL17 and CCL22 expression in bone marrow-derived dendritic cells (BMDCs) from wild-type and S1pr2 gene-deficient mice. Addition of IL-4 (10 ng/mL) markedly increased the levels of CCL17 and CCL22, but IL-4-induced CCL17 and CCL22 expression was significantly blunted in BMDCs from S1pr2 gene-deficient mice. Furthermore, pretreatment with JTE-013 (1-30 μM) dose-dependently suppressed this induction in BMDCs from wild-type mice. Our results demonstrate that blockage of S1P₂ ameliorates not only DNCB-induced atopic dermatitis symptoms but also Th2 cell-attracting capacity of dendritic cells, suggesting S1P₂ as a potential therapeutic target for atopic dermatitis.

Keywords: S1pr2 gene-deficient mice; atopic dermatitis; bone marrow-derived dendritic cell; cytokines; sphingosine-1-phosphate; sphingosine-1-phosphate receptor type 2 (S1P2).

Forsythiae Fructus, Lonicerae Flos, and Scutellariae Radix are medicinal herbs that possess anti-inflammatory and anti-atopic effects. Hence, we investigated the effects of a mixture (ADM), containing arctigenin, hederagenin, and baicalein, which are the main compound from these herbs on atopic dermatitis (AD) skin lesions and the underlying molecular mechanisms. ADM was topically applied to dorsal skin lesions of 2,4-dinitrochlorobenzene- (DNCB-) induced ICR mice, and the expressions of proinflammatory mediators and HPA axis hormones were investigated. The topical application of 0.5% ADM significantly reduced the DNCB-induced symptoms of AD in ICR mice. Histological analysis showed that ADM exerted antiatopic effects by reducing the epidermal thickness and mast cell infiltration into skin lesions. 0.5% ADM attenuated the levels of TNF-α, IFN-γ, IL-4, and VEGF in skin lesions and serum IgE. The production of Th1-/Th2-related cytokines in splenic tissues, including TNF-α, IFN-γ, IL-12, and IL-4, were also decreased by ADM treatment. ADM diminished corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosteroid (CORT) production in DNCB-induced mice. In vitro, ADM reduced the productions of TARC/CCL17, MDC/CCL22, IL-6, and ICAM-1 in TNF-α/IFN-γ- (TI-) stimulated HaCaT cells by suppressing the ERK and JNK signaling pathways. In addition, ADM inhibited corticotropin-releasing hormone/substance P- (CRH/SP-) induced VEGF production in HMC-1 cells. These results suggest that ADM may have therapeutic potential in AD by reducing inflammation and attenuating HPA axis activation.

Aim: Leishmania donovani, the causative agent for visceral leishmaniasis (VL), modulates host monocytes-macrophages to ensure its survival. However, knowledge regarding the host-parasite interactions underpinning the disease remains limited. As disease progression is associated with polarization of monocytes/macrophages towards M2 which is regulated by cytokines IL-4/IL-13 and IL-10, this study evaluated the status of key IL-4 and IL-10 driven markers in experimental models of VL, as also evaluated their correlation, if any, with parasite load.

Methods: In liver and splenic tissues from L.donovani infected hamsters and BALB/c mice, the parasite burden was determined along with mRNA expression of IL-4 driven markers i.e. CD206, Arginase-I, CCL17, CCL22, PPARγ , STAT-6,KLF-4, FIZZ1 and YM1 along with IL-10 driven markers, CXCL13, IL-10, TGFβ, VDR,CCR2 and CYP27A1.

Results: The mRNA expression of IL-4 and IL-10 driven markers was enhanced in both models, but only in the hamster model, the splenic tissues demonstrated a positive correlation between all the IL-10 driven markers and parasite load.

Conclusions: Contrary to human VL, both models demonstrated an increased expression of IL-4 and IL-10 driven markers.

Keywords: Animal models; IL-4 and IL-10 driven gene expression; Visceral Leishmaniasis; monocytes/macrophages.

Background: Polymyositis/dermatomyositis (PM/DM) is an autoimmune disease that is sometimes complicated with rapidly progressive interstitial lung disease (RPILD). However, serum and lung biomarkers that can predict RPILD development remain unclear.

Objectives: To determine potential serum and lung biomarkers that can predict RPILD development in patients with PM/DM-ILD.

Methods: In total, 49 patients with PM/DM-ILD were enrolled. We measured the serum levels of 41 cytokines/chemokines, ferritin and anti-MDA5 antibody, compared them between the RPILD (n = 23) and non-RPILD (n = 26) groups, and ranked them by their importance through random forest analysis. To distinguish the two groups, we determined biomarker combinations by logistic regression analysis. We also measured the bronchoalveolar lavage fluid (BALF) levels of 41 cytokines/chemokines. Using immunohistochemistry, we examined IL-15 expression in lung tissues. The IL-15 production was also investigated using A549 and BEAS-2B cells.

Results: The RPILD group had significantly higher IL-15, IL-1RA, IL-6, CXCL10, VCAM-1, anti-MDA5 antibody and ferritin serum levels than the non-RPILD group, but it had a significantly low CCL22 level. Meanwhile, anti-MDA5 antibody, IL-15, CXCL8, CCL22, IL-1RA and ferritin were the best combination to distinguish the two groups. IL-15 and CCL22 were also predictive marker for RPILD development in anti-MDA5 antibody-positive patients. Additionally, the RPILD group had significantly high IL-15 levels in BALF. The lung tissues expressed IL-15, which increased after cytokine stimulation in the A549 cells.

Conclusion: This study identified a combination of biomarkers predicting PM/DM-RPILD progression, and IL-15 is an important cytokine for predicting RPILD development and reflecting ILD severity.

Keywords: anti-MDA5 antibody; biomarkers; dermatomyositis; interleukin-15; polymyositis; rapidly progressive interstitial lung disease.

BACKGROUND

Recently, evidence has been obtained to suggest that inflammation is provoked through upregulation of macrophage migration inhibitory factor (MIF) expression by steroids. However, little is known regarding the effect of steroids on MIF expression in human keratinocytes and the counter-effect of epigallocatechin-3-gallate (EGCG), a member of the class of green tea polyphenols.

OBJECTIVE

We determined whether or not steroids cause the upregulation of MIF in human keratinocytes, and if so, whether or not EGCG suppresses MIF upregulation in keratinocytes by steroids. We then assessed the effects of EGCG on MIF-induced Th-related chemokine and cytokine expression in keratinocytes.

METHODS

HaCaT keratinocytes were first treated with dexamethasone in the presence or absence of EGCG in the culture medium. The keratinocytes were then treated with recombinant human (rh)-MIF in the presence or absence of EGCG in the culture medium. The expression of mRNA and protein in Th-related cytokines and chemokines, including MIF in the keratinocytes, was measured by real-time reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay.

RESULTS

Dexamethasone significantly enhanced MIF expression in human keratinocytes, and EGCG significantly downregulated the expression of dexamethasone-induced MIF. EGCG also significantly downregulated rh-MIF-induced expression of Th-related cytokines and chemokines, such as interleukin (IL)-6, IL-18, transforming growth factor-β, CCL17, CCL22 and CXCL10, in human keratinocytes.

CONCLUSIONS

These results demonstrated that EGCG may have a novel pharmacological effect to prevent steroid-induced tachyphylaxis and inflammation by suppressing the expression of MIF in human keratinocytes.

Objective: This study set out to explore the regulatory mechanism of miR-130a-5p in cisplatin (DDP)-resistant gastric cancer (GC) cells.

Materials and methods: Forty cases of GC and paracancerous tissues were collected, and the miR-130a-5p and CCL22 levels were detected by qRT-PCR. DDP-resistant cell lines of GC cells were established. Cell viability, invasion, and apoptosis were measured by CCK-8, Transwell, and flow cytometry, respectively. The relationship between miR-130a-5p and CCL22 was verified by dual-luciferase reporter enzyme, and the protein levels of caspase-3, bax, bcl-2, and CCL22 were determined by Western blot.

Results: miR-130a-5p was low expressed in GC tissues and cells, while CCL22 showed marked negative correlation, and the area under the curve (AUC) for diagnosing GC was not less than 0.850. Up-regulating miR-130a-5p or knocking down CCL22 expression can inhibit the proliferation and invasion of GC cells and promote their apoptosis, reverse the resistance of NCI-N87/DDP to DDP, and also enhance the chemosensitivity of GC cells. Dual-luciferase reporter enzyme identified that there was a targeted relationship between miR-130a-5p and CCL22. At the same time, miR-130a-5p and CCL22 were up-regulated or down-regulated, and the malignant proliferation, invasion, apoptosis, and DDP chemotherapy resistance of the cells had no difference compared with miR-NC with transfection-unrelated sequences.

Conclusion: Up-regulating miR-130a-5p can enhance the sensitivity of DDP-resistant GC cells to chemotherapy and regulate their biological function by targeted inhibition of CCL22.

Keywords: CCL22; chemosensitivity; cisplatin; gastric cancer; miR-130a-5p.

Asthmatics sensitized to fungi are reported to have more severe asthma, yet the immunopathogenic pathways contributing to this severity have not been identified. In a pilot assessment of human asthmatics, those subjects sensitized to fungi demonstrated elevated levels of the common γ-chain cytokine IL-7 in lung lavage fluid, which negatively correlated with the lung function measurement PC20. Subsequently, we show that IL-7 administration during experimental fungal asthma worsened lung function and increased the levels of type 2 cytokines (IL-4, IL-5, IL-13), proallergic chemokines (CCL17, CCL22) and proinflammatory cytokines (IL-1α, IL-1β). Intriguingly, IL-7 administration also increased IL-22, which we have previously reported to drive immunopathogenic responses in experimental fungal asthma. Employing IL22CreR26ReYFP reporter mice, we identified γδ T cells, iNKT cells, CD4 T cells and ILC3s as sources of IL-22 during fungal asthma; however, only iNKT cells were significantly increased after IL-7 administration. IL-7-induced immunopathogenesis required both type 2 and IL-22 responses. Blockade of IL-7Rα in vivo resulted in attenuated IL-22 production, lower CCL22 levels, decreased iNKT cell, CD4 T-cell and eosinophil recruitment, yet paradoxically increased dynamic lung resistance. Collectively, these results suggest a complex role for IL-7 signaling in allergic fungal asthma.

Following long‑term exposure to endotoxins, macrophages enter an immunosuppressive state that renders them unable respond to subsequent exposures to endotoxin, a phenomenon that is termed 'endotoxin tolerance'. Endotoxin tolerance increases the risks of secondary infection and mortality in patients with sepsis. In endotoxin‑tolerant macrophages, the mixed variation of gene transcription is referred to as macrophage reprogramming. The mechanisms underlying macrophage reprogramming remain unclear at present. Interferon‑induced double‑stranded RNA‑dependent protein kinase (PKR) is a widely expressed serine/threonine protein kinase. In addition to antiviral effects, PKR regulates the transcription of inflammatory cytokines by affecting transcription factors. However, the role of PKR in macrophage reprogramming remains to be elucidated. In the present study, the expression of inflammatory cytokines differed in lipopolysaccharide (LPS)‑tolerant RAW264.7 macrophages compared with LPS‑activated macrophages. Specifically, reverse transcription‑quantitative polymerase chain reaction results demonstrated that the mRNA levels of tumor necrosis factor‑α, interleukin‑1β (IL‑1β), C‑X‑C motif chemokine ligand 11, C‑C motif chemokine ligand (CCL17), CCL22 and suppressor of cytokine signaling 3 were decreased, and mRNAs levels of arginase‑1 (Arg1) and nitric oxide synthase 2 (iNOS) were increased, in LPS‑tolerant macrophages compared with LPS‑activated macrophages. Furthermore, western blot analysis demonstrated that the protein levels of phosphorylated (p)‑PKR were significantly decreased in the LPS‑tolerant cells. PKR activation with rotenone (10 µM) abrogated endotoxin tolerance by increasing the levels of the IL‑1β, CCL17 and CCL22 mRNAs and decreasing the levels of the Arg1 and iNOS mRNAs. Furthermore, western blotting demonstrated that AKT was markedly inactivated in endotoxin‑tolerant cells, as indicated by reduced p‑AKT levels. However, levels of p‑AKT were markedly increased following rotenone‑induced PKR activation in endotoxin‑tolerant cells. Ly294002 (10 µM), a phosphatidylinositol‑4,5‑bisphosphate 3‑kinase (PI3K)/AKT signaling inhibitor, partially reversed the rotenone‑induced alleviation of endotoxin tolerance. These results demonstrated that PKR inhibition mediated endotoxin tolerance in macrophages, and these effects were partially mediated by PI3K/AKT signaling. PKR may be a potential target for the treatment of endotoxin tolerance in patients with sepsis.

Minocycline/tetracycline is clinically used for the treatment of bullous pemphigoid (BP), and its clinical benefits are superior to those of prednisolone when considering adverse events. Although the clinical benefits of minocycline/tetracycline are well known, its immunosuppressive mechanisms are still unclear. In this study, we investigated the immunomodulatory effects of traditional anti-BP drugs (minocycline, nicotinic acid amide, dexamethasone and cyclosporine) on CD163+ M2 macrophages in vitro, with special focus on the production of CCL18 and CCL22, both of which are produced by CD163+ M2 macrophages in the lesional skin of BP and are increased in the serum of BP patients. Minocycline decreased the production of CCL22, CCL24 and CCL26 as well as CCL2 from M2 macrophages. CCL18 from M2 macrophages was decreased by dexamethasone and cyclosporine, but not decreased by minocycline. These data suggest that the clinical benefit of minocycline is partially explained by its suppressive effects against the production of specific Th2 chemokines from M2 macrophages, which should contribute to the recruitment of Th2 cells and eosinophils in the lesional skin of BP patients.

The role of fibroblasts in tissue fibrosis has been extensively studied. Activated fibroblasts, namely myofibroblasts, produce pathological extracellular matrix. CD248, a type I transmembrane glycoprotein, is expressed in fibroblasts after birth. In human chronic kidney disease, upregulated CD248 in myofibroblasts is linked to poor renal survival. In this study, we demonstrated a novel interaction between CD248 and macrophages to be a key step in mediating tissue fibrosis. CD248 was upregulated in myofibroblasts in murine models of renal and peritoneal fibrosis. Cd248 knockout (Cd248^-/-) could attenuate both renal and peritoneal fibrosis. By parabiosis of GFP reporter mice and Cd248^-/- mice, we showed that attenuation of renal fibrosis was associated with a decrease of macrophage infiltration in Cd248^-/- mice. Moreover, decrease of chemokine (C-C motif) ligand 17 and Ccl22 was found in macrophages isolated from the fibrotic kidneys of Cd248^-/- mice. Because galectin-3-deficient macrophages showed decreased Ccl17 and Ccl22 in fibrotic kidneys, we further demonstrated that CD248 interacted specifically with galectin-3 of macrophages who then expressed CCL17 to activate collagen production in myofibroblasts. Mice with DNA vaccination targeting CD248 showed decreased fibrosis. We thus propose that CD248 targeting should be studied in the clinical tissue fibrosis setting.

Several lines of research support immune system dysregulation in psychotic disorders. However, it remains unclear whether the immunological marker alterations are stable and how they associate with brain glial cell function. This longitudinal study aimed at investigating whether peripheral immune functions are altered in the early phases of psychotic disorders, whether the changes are associated with core symptoms, remission, brain glial cell function, and whether they persist in a one-year follow-up. Two independent cohorts comprising in total of 129 first-episode psychosis (FEP) patients and 130 controls were assessed at baseline and at the one-year follow-up. Serum cyto-/chemokines were measured using a 38-plex Luminex assay. The FEP patients showed a marked increase in chemokine CCL22 levels both at baseline (p < 0.0001; Cohen's d = 0.70) and at the 12-month follow-up (p = 0.0007) compared to controls. The group difference remained significant (p = 0.0019) after accounting for relevant covariates including BMI, smoking, and antipsychotic medication. Elevated serum CCL22 levels were significantly associated with hallucinations (ρ = 0.20) and disorganization (ρ = 0.23), and with worse verbal performance (ρ = -0.23). Brain glial cell activity was indexed with positron emission tomography and the translocator protein radiotracer [¹¹C]PBR28 in subgroups of 15 healthy controls and 14 FEP patients with serum CCL22/CCL17 measurements. The distribution volume (V_T) of [¹¹C]PBR28 was lower in patients compared to controls (p = 0.026; Cohen's d = 0.94) without regionally specific effects, and was inversely associated with serum CCL22 and CCL17 levels (p = 0.036). Our results do not support the over-active microglia hypothesis of psychosis, but indicate altered CCR4 immune signaling in early psychosis with behavioral correlates possibly mediated through cross-talk between chemokine networks and dysfunctional or a decreased number of glial cells.

Clinical and experimental evidence supports a role for cardiac glycosides (CGs) as potential novel anticancer drugs. However, there are no studies reporting the effect of CGs on the inflammatory tumor microenvironment (TME), which plays a central role in tumor progression and invasiveness. We investigated whether digitoxin affects a) specific pathways involved in motility and/or activation of different cell types shaping TME, and b) cancer cell growth and invasiveness in response to TME-associated factors. To test our hypothesis, conditioned media (CM) from polarized macrophages, and apoptotic or non-apoptotic ovarian cancer cells (SKOV3) were tested as chemoattractants for endothelial cells, monocytes and cancer cells. We demonstrated that CM from M1 (LPS/IFNγ) and M2 (IL-4/IL-13) polarized macrophages, which mimic inflammatory TME, increased both HUVEC migration and tubularization. Treatment of HUVECs with digitoxin at concentrations within its plasma therapeutic range counteracted these effects. Digitoxin affected the expression of neither M1 (CD80/CD68) nor M2 (CD206/CD163) activation markers, nor the amount of cell-bound IL-1β and CCL22. Accordingly, HUVEC migration in response to CM from digitoxin-treated activated macrophages was unchanged. These data point to a direct effect of digitoxin on HUVEC signaling rather than on the modulation of the cytokine profile released from activated macrophages. At variance with what observed for HUVECs, digitoxin did not prevent monocyte migration induced by SKOV3 CM. In addition, digitoxin significantly impaired SKOV3 growth and migration in response to M1 or M2 macrophage CM. Finally, we showed that digitoxin inhibited FAK phosphorylation in SKOV3 but not PYK2 phosphorylation in monocytes, thus providing a molecular mechanism accounting for the observed differential anti-migratory effect. Overall, digitoxin counteracted salient features of the inflammatory ovarian cancer microenvironment, laying the ground for potential digitoxin repositioning as an anticancer drug.

OBJECTIVE

In rheumatoid arthritis (RA), it is of major importance to identify non-responders to tumour necrosis factor-α inhibitors (TNFi) before starting treatment, to prevent a delay in effective treatment. We developed a protein score for the response to TNFi treatment in RA and investigated its predictive value.

METHODS

In RA patients eligible for biological treatment included in the BiOCURA registry, 53 inflammatory proteins were measured using xMAP® technology. A supervised cluster analysis method, partial least squares (PLS), was used to select the best combination of proteins. Using logistic regression, a predictive model containing readily available clinical parameters was developed and the potential of this model with and without the protein score to predict European League Against Rheumatism (EULAR) response was assessed using the area under the receiving operating characteristics curve (AUC-ROC) and the net reclassification index (NRI).

RESULTS

For the development step (n = 65 patient), PLS revealed 12 important proteins: CCL3 (macrophage inflammatory protein, MIP1a), CCL17 (thymus and activation-regulated chemokine), CCL19 (MIP3b), CCL22 (macrophage-derived chemokine), interleukin-4 (IL-4), IL-6, IL-7, IL-15, soluble cluster of differentiation 14 (sCD14), sCD74 (macrophage migration inhibitory factor), soluble IL-1 receptor I, and soluble tumour necrosis factor receptor II. The protein score scarcely improved the AUC-ROC (0.72 to 0.77) and the ability to improve classification and reclassification (NRI = 0.05). In validation (n = 185), the model including protein score did not improve the AUC-ROC (0.71 to 0.67) or the reclassification (NRI = -0.11).

CONCLUSIONS

No proteomic predictors were identified that were more suitable than clinical parameters in distinguishing TNFi non-responders from responders before the start of treatment. As the results of previous studies and this study are disparate, we currently have no proteomic predictors for the response to TNFi.

Neuropathic pain is a chronic condition that remains a major clinical problem owing to high resistance to available therapy. Recent studies have indicated that chemokine signaling pathways are crucial in the development of painful neuropathy; however, the involvement of CC chemokine receptor 4 (CCR4) has not been fully elucidated thus far. Therefore, the aim of our research was to investigate the role of CCR4 in the development of tactile and thermal hypersensitivity, the effectiveness of morphine/buprenorphine, and opioid-induced tolerance in mice exposed to chronic constriction injury (CCI) of the sciatic nerve. The results of our research demonstrated that a single intrathecal or intraperitoneal administration of C021, a CCR4 antagonist, dose dependently diminished neuropathic pain-related behaviors in CCI-exposed mice. After sciatic nerve injury, the spinal expression of CCL17 and CCL22 remained unchanged in contrast to that of CCL2, which was significantly upregulated until day 14 after CCI. Importantly, our results provide evidence that in naive mice, CCL2 may evoke pain-related behaviors through CCR4 because its pronociceptive effects are diminished by C021. In CCI-exposed mice, the pharmacological blockade of CCR4 enhanced the analgesic properties of morphine/buprenorphine and delayed the development of morphine-induced tolerance, which was associated with the silencing of IBA-1 activation in cells and decrease in CCL2 production. The obtained data suggest that the pharmacological blockade of CCR4 may be a new potential therapeutic target for neuropathic pain polytherapy.

Keywords: CCL17; CCL2; CCL22; buprenorphine; chemokines; mice; morphine; opioids.

Chronic skin inflammatory diseases, such as atopic dermatitis, are associated with a dysfunctional skin barrier due to an increase in various inflammatory stimuli, for instance inflammatory cytokines and chemokines. In particular, CCL17 and CCL22 expression is increased in patients with chronic skin inflammation. In this study, we synthesized several α- and β-anomers of dihydroergosterol (DHE)-glycosides and assessed their effects on CCL17 and CCL22 expression. We confirmed that the β-anomers of DHE-glycosides were superior to α-anomers of DHE-glycosides in inhibiting CCL17 and CCL22 mRNA and protein expression. In addition, we determined that DHE-glycoside β-anomers showed strong inhibitory activity towards pro-inflammatory cytokine mRNA and protein expression, including that of TNF-α, IL-6, and IL-1β- in stimulated HaCaT cells. These results imply that DHE-glycoside α- and β-anomers should be separated during synthesis of drugs for chronic skin inflammation. Our results also suggest that β-anomers of DHE-glycosides may play an important role as new drugs for chronic skin inflammation because of their ability to inhibit the skin inflammatory biomarker proteins CCL17 and CCL22.

Purpose: Radiation therapy elicits profound alterations in gene expression in tumor cells. This study aims to determine the dynamic changes in the expression of immunity-associated genes in nasopharyngeal carcinoma (NPC) cells upon radiation therapy.

Methods and materials: The study was performed using NPC patient-derived tumor xenograft tumors, cell lines, CCR4⁺ CD8 T cells sorted from peripheral blood mononuclear cells of healthy volunteers, and TCGA-derived bulk RNA-seq or single-cell RNA-seq (scRNA-seq) data sets. Patient-derived tumor xenograft tumors or cell lines were irradiated and collected for bulk RNA sequencing or for CCL22 expression and release detection. Malignant phenotypes and radiosensitivity were assessed in cells with or without overexpression of CCL22 or recombinant CCL22 treatment in the presence or absence of irradiation. TCGA data sets were used for uncovering CCR4 status in subtypes of T cells. CCL22 in supernatants, cell lysates, or serum samples was measured with enzyme-linked immunosorbent assay.

Results: CCL22 was significantly increased in the irradiated patient-derived tumor xenograft tumors, the supernatants and cell lysates collected from irradiated NPC cell lines, and the serum of patients who received radiation therapy. No alterations of malignant phenotypes were found in tumor cells with CCL22 overexpression or recombinant CCL22 treatment. Kaplan-Meier analysis revealed that CCL22 or its receptor CCR4 positively correlated with cytotoxic T lymphocyte signatures, and high expression of CCL22 or CCR4 was associated with better prognosis for patients with NPC. scRNA-seq data set-based analysis demonstrated that CCR4 was expressed in multiple subtypes of T cells, including effector CD8 T cells. Chemotaxis assay indicated that CCR4⁺ CD8 T cells could be recruited by CCL22 treatment.

Conclusion: The radiation-enhanced release of CCL22 from NPC cells promotes migration of CCR4 ⁺ effector CD8 T cells, which might partially be associated with radiation therapy-mediated antitumor immunity.

Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.

Objective: To identify candidate biomarkers associated with neuromyelitis optica spectrum disorder (NMOSD) using high-throughput technologies that broadly assay the concentrations of serum analytes and frequencies of immune cell subsets.

Methods: Sera, peripheral blood mononuclear cells (PBMCs), and matched clinical data from participants with NMOSD and healthy controls (HCs) were obtained from the Collaborative International Research in Clinical and Longitudinal Experience Study NMOSD biorepository. Flow cytometry panels were used to measure the frequencies of 39 T-cell, B-cell, regulatory T-cell, monocyte, natural killer (NK) cell, and dendritic cell subsets in unstimulated PBMCs. In parallel, multiplex proteomics assays were used to measure 46 serum cytokines and chemokines in 2 independent NMOSD and HC cohorts. Multivariable regression models were used to assess molecular and cellular profiles in NMOSD compared with HC.

Results: NMOSD samples had a lower frequency of CD16⁺CD56⁺ NK cells. Both serum cohorts and multivariable logistic regression revealed increased levels of B-cell activating factor associated with NMOSD. Interleukin 6, CCL22, and CCL3 were also elevated in 1 NMOSD cohort of the 2 analyzed. Multivariable linear regression of serum analyte levels revealed a correlation between CX3CL1 (fractalkine) levels and the number of days since most recent disease relapse.

Conclusions: Integrative analyses of cytokines, chemokines, and immune cells in participants with NMOSD and HCs provide congruence with previously identified biomarkers of NMOSD and highlight CD16⁺CD56⁺ NK cells and CX3CL1 as potential novel biomarker candidates.

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127-/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation.

Keywords: cytokine; enterovirus; regulatory T cell; rhinovirus; type 1 diabetes.

Macrophages are highly plastic cells, responding to diverse environmental stimuli to acquire different functional phenotypes. Signaling through MAPKs has been reported to regulate the differentiation of macrophages, but the role of ERK5 in IL-4-mediated M2 macrophage differentiation is still unclear. Here, we showed that the ERK5 signaling pathway plays a critical role in IL-4-induced M2 macrophage differentiation. Pharmacologic inhibition of MEK5, an upstream activator of ERK5, markedly reduced the expression of classical M2 markers, such as Arg-1, Ym-1, and Fizz-1, as well as the production of M2-related chemokines and cytokines, CCL22, CCL17, and IGF-1 in IL-4-stimulated macrophages. Moreover, pharmacologic inhibition of ERK5 also decreased the expression of several M2 markers induced by IL-4. In accordance, myeloid cell-specific Erk5 depletion (Erk5^∆mye ), using LysM^cre /Erk5^f/f mice, confirmed the involvement of ERK5 in IL-4-induced M2 polarization. Mechanistically, the inhibition of ERK5 did not affect STAT3 or STAT6 phosphorylation, suggesting that ERK5 signaling regulates M2 differentiation in a STAT3 and STAT6-independent manner. However, genetic deficiency or pharmacologic inhibition of the MEK5/ERK5 pathway reduced the expression of c-Myc in IL-4-activated macrophages, which is a critical transcription factor involved in M2 differentiation. Our study thus suggests that the MEK5/ERK5 signaling pathway is crucial in IL-4-induced M2 macrophage differentiation through the induction of c-Myc expression.

Keywords: ERK5; IL-4; M2 macrophages; MEK5; macrophage polarization.