Distinct Cytokine Profiles Research Articles

Dermal IRF4+ dendritic cells and monocytes license CD4+ T helper cells to distinct cytokine profiles

Antigen (Ag)-presenting cells (APC) instruct CD4+ helper T (Th) cell responses, but it is unclear whether different APC subsets contribute uniquely in determining Th differentiation in pathogen-specific settings. Here, we use skin-relevant, fluorescently-labeled bacterial, helminth or fungal pathogens to track and characterize the APC populations that drive Th responses in vivo. All pathogens are taken up by a population of IRF4+ dermal migratory dendritic cells (migDC2) that similarly upregulate surface co-stimulatory molecules but express pathogen-specific cytokine and chemokine transcripts. Depletion of migDC2 reduces the amount of Ag in lymph node and the development of IFNγ, IL-4 and IL-17A responses without gain of other cytokine responses. Ag+ monocytes are an essential source of IL-12 for both innate and adaptive IFNγ production, and inhibit follicular Th cell development. Our results thus suggest that Th cell differentiation does not require specialized APC subsets, but is driven by inducible and pathogen-specific transcriptional programs in Ag+ migDC2 and monocytes.

Distinct circulating cytokine levels in patients with angiography-proven coronary artery disease compared to disease-free controls

Abstract Background Systemic inflammation has a critical role in the development of coronary artery disease (CAD). Identification of inflammatory pathways may provide a platform for novel therapeutic approaches. Purpose We sought to determine whether there are differences in circulating cytokine profiles between patients with CAD and disease-free controls. Methods Study population consisted of 458 patients who underwent diagnostic invasive coronary angiography for clinical indications. Altogether 312 patients had angiographically significant CAD whereas 146 had no angiographically-detected coronary atherosclerosis. We measured the serum concentrations of 48 circulating cytokines. Results Patients with CAD had increased levels of interleukin-1 receptor antagonist (IL-1-RA), interleukin-2 receptor alpha (IL-2Rα), IL-3, IL-4, IL-6, IL-9, IL-10, IL-16, IL-17, IL-18, granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), macrophage inflammatory protein-1-beta (MIP-1-β), tumor necrosis factor-alpha (TNF-α), TNF-β, cutaneous T-cell-attracting chemokine (CTACK), growth regulated oncogene alpha (GRO-α), hepatocyte growth factor (HGF), interferon alpha-2 (IFNα-2), leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), macrophage migration inhibitory factor (MIF), monokine induced by gamma interferon (MIG), beta-nerve growth factor (β-NGF), stem cell factor (SCF) and stromal cell-derived factor 1 alfa (SDF1α). On a logistic multivariate regression model adjusted with age, sex, hypertension and treatment for diabetes, increased levels of IL-4 (p=0.027, OR 1.090), IL-9 (p=0.000, OR 1.013), IL-17 (p=0.011, OR 1.005), CTACK (p=0.008, OR 1.001), MIP-1-β (p=0.004, OR 1.006), GRO-α (p=0.008, OR 1.004), TNF-α (p=0.019, OR 1.011) were independently associated with atherosclerosis. Conclusions Patients with CAD have distinct circulating cytokine profiles compared to disease-free controls. Based on these findings, certain cytokines may have a pivotal role in the development of atherosclerotic cardiovascular disease and cytokine-mediated pathway appear as a promising target for cardiovascular drug development. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Finnish Medical Foundation, Helsinki, Finland; The Finnish Foundation for Cardiovascular Research, Helsinki, Finland

Characterizing the endometrial fluid cytokine profile in women with endometriosis.

To compare growth factor and cytokine profiles in the endometrial secretions of patients with and without endometriosis to determine whether a particular protein profile is predictive of the disease. Patients undergoing laparoscopic gynecologic surgery for benign indications were recruited for this prospective cohort study. Prior to surgery, endometrial fluid was aspirated and multiplex immunoassay was used to quantify 7 cytokines and growth factors. During surgery, each patient was staged according to the ASRM staging system for endometriosis. Cytokines and growth factors were evaluated using the Mann-Whitney and Kruskal-Wallis tests. Combinations of cytokines were evaluated using logistic regression analysis, and ROC curves were generated to evaluate the predictive capacity of the assay. Endometrial secretions were analyzed from 60 patients. Nineteen had stage 3-4 endometriosis, 19 had stage 1-2 disease, and 22 had no endometriosis. There were no significant differences between controls and stage 1-2 endometriosis; however, levels of IL-1α and IL-6 were significantly increased in women with moderate-to-severe disease. A combination of IL-1α, IL-1β, and IL-6 in endometrial secretions predicts stage 3-4 endometriosis with an AUC of 0.78. A threshold value of 118pg/mL yields a sensitivity of 75% and specificity of 70%. Aspiration of endometrial fluid is a safe and effective approach for evaluating the endometrial profile of women with endometriosis. Women with moderate-to-severe endometriosis demonstrate a distinct cytokine profile compared to controls. A combination of IL-1α, IL-1β, and IL-6 in the endometrial secretions is predictive of stage 3-4 endometriosis, but is not predictive of minimal-to-mild disease.

Decreased Frequency of Intestinal CD39+ γδ+ T Cells With Tissue-Resident Memory Phenotype in Inflammatory Bowel Disease.

The ectoenzymes CD39 and CD73 play a major role in controlling tissue inflammation by regulating the balance between adenosine triphosphate (ATP) and adenosine. Still, little is known about the role of these two enzymes and ATP and its metabolites in the pathophysiology of inflammatory bowel disease (IBD). We isolated mononuclear cells from peripheral blood and lamina propria of the large intestine of patients diagnosed with IBD and of healthy volunteers. We then comprehensively analyzed the CD39 and CD73 expression patterns together with markers of activation (HLA-DR, CD38), differentiation (CCR7, CD45RA) and tissue-residency (CD69, CD103, CD49a) on CD4+, CD8+, γδ+ T cells and mucosa-associated invariant T cells using flow cytometry. CD39 expression levels of γδ+ and CD8+ T cells in lamina propria lymphocytes (LPL) were much higher compared to peripheral blood mononuclear cells. Moreover, the frequency of CD39+ CD4+ and CD8+, but not γδ+ LPL positively correlated with T-cell activation. The frequency of CD39+ cells among tissue-resident memory LPL (Trm) was higher compared to non-Trm for all subsets, confirming that CD39 is a marker for the tissue-resident memory phenotype. γδ+ Trm also showed a distinct cytokine profile upon stimulation – the frequency of IFN-γ+ and IL-17A+ cells was significantly lower in γδ+ Trm compared to non-Trm. Interestingly, we observed a decreased frequency of CD39+ γδ+ T cells in IBD patients compared to healthy controls (p = 0.0049). Prospective studies need to elucidate the exact role of this novel CD39+ γδ+ T-cell population with tissue-resident memory phenotype and its possible contribution to the pathogenesis of IBD and other inflammatory disorders.

Is There a Role for Basophils in Cancer?

Basophils were identified in human peripheral blood by Paul Ehrlich over 140 years ago. Human basophils represent <1% of peripheral blood leukocytes. During the last decades, basophils have been described also in mice, guinea pigs, rabbits, and monkeys. There are many similarities, but also several immunological differences between human and mouse basophils. There are currently several strains of mice with profound constitutive or inducible basophil deficiency useful to prove that these cells have specific roles in vivo. However, none of these mice are solely and completely devoid of all basophils. Therefore, the relevance of these findings to humans remains to be established. It has been known for some time that basophils have the propensity to migrate into the site of inflammation. Recent observations indicate that tissue resident basophils contribute to lung development and locally promote M2 polarization of macrophages. Moreover, there is increasing evidence that lung-resident basophils exhibit a specific phenotype, different from circulating basophils. Activated human and mouse basophils synthesize restricted and distinct profiles of cytokines. Human basophils produce several canonical (e.g., VEGFs, angiopoietin 1) and non-canonical (i.e., cysteinyl leukotriene C4) angiogenic factors. Activated human and mouse basophils release extracellular DNA traps that may have multiple effects in cancer. Hyperresponsiveness of basophils has been demonstrated in patients with JAK2V617F-positive polycythemia vera. Basophils are present in the immune landscape of human lung adenocarcinoma and pancreatic cancer and can promote inflammation-driven skin tumor growth. The few studies conducted thus far using different models of basophil-deficient mice have provided informative results on the roles of these cells in tumorigenesis. Much more remains to be discovered before we unravel the hitherto mysterious roles of basophils in human and experimental cancers.

Comparison of Expression Levels of miR-29b-3p and miR-326 in T Helper-1 and T Helper-17 Cells Isolated from Responsive and Non-responsive Relapsing-remitting Multiple Sclerosis Patients Treated with Interferon-beta.

T helper type 1 (Th1) and Th17 Cells with distinct cytokine profiles including interferon-gamma (IFN-γ) and interleukin 17 (IL-17) have a pivotal role in neuroinflammation and myelin destruction in the central nervous system (CNS) in MS. MicroRNA-29b (MiR-29b) and miR-326 contribute to regulating Th1 and Th17 differentiation and altered expression of the miRNAs could be associated with response to treatment in multiple sclerosis (MS). Therefore, our study aimed to evaluate the percentage of Th1 and Th17 and determining the expression levels of miR-29b-3p and miR-326 in these lymphocyte subpopulations between responsive and non-responsive to interferon beta (IFN-β) therapy in relapsing-remitting multiple sclerosis (RRMS) patients. The present study was performed on 40 RRMS patients following treatment with IFN-β. The percentage of Th1 cells and Th17 cells were determined by flow cytometry in responsive and non-responsive patients. The expression levels of miR-29b-3p and miR-326 were assessed in Th1 and Th17 cells by quantitative polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the plasma levels of IFN-γ and IL-17A. No significant difference was observed in the percentage of Th1 and Th17 cells as well as the expression levels of miR-29b-3p and miR-326 (in Th1 and Th17, respectively) in treated patients. Also, we did not find any significant difference in IFN-γ and IL-17A plasma concentration between responsive or non-responsive to IFN-β therapy in patients with RRMS. IFN-β may regulate other miRNAs in Th1 and Th17 cells than miR29b-3p and miR-326 in MS patients.

Abstract 4231: Factors predictive of CAR T cell associated hemophagocytic lymphohistiocytosis (HLH)

Introduction: HLH is a T cell mediated inflammatory syndrome associated with immune activation and NK cell dysfunction. In the context of CAR T cells, HLH occurs in a subset of those with cytokine release syndrome (CRS), yet predisposing factors are not yet well established. Utilizing our ongoing phase I study of CD22/4-1BB CAR T cells in children and young adults with CD22+ B cell malignancies (NCT02315612), we evaluated patient and product characteristics to elucidate factors associated with HLH. Methods: We retrospectively reviewed multiple variables to identify correlates of HLH in those infused. Definitions for CRS and HLH are in Table 1. Multiple univariate analyses were performed to identify risk factors for HLH; variables with significance were incorporated into a multiple logistic regression model to predict those at risk of HLH-like CRS. Results: 52 of 60 (86.7%) treated subjects developed CRS; 21 (40.4%) of whom subsequently developed HLH. The following factors were associated with HLH: lower baseline blood and marrow NK% and higher CD3% cells; CD4/CD8 T cell selection (TCS) of the apheresis product (which increased CD3 and reduced NK cell populations); and higher IL18. Following infusion, distinct cytokine profiles and prolonged predominance of CD8 T cells distinguished those with HLH from those without it. Multiple logistic regression identified a) use of TCS, b) higher grade CRS and c) high baseline bone marrow T/NK ratio as most predictive of HLH; leading to a model with 75% sensitivity and 80% specificity. Conclusion: Our results provide novel insights into factors associated with CAR T cell related HLH and implicate preexisting T and NK cell populations, baseline cytokines, apheresis selection methodologies and CAR T cell expansion parameters in the pathophysiology. With a goal of earlier intervention to prevent HLH, the predictive model will be prospectively evaluated. Biologic correlative studies and assessment of generalizability in alternate CAR T cell constructs are planned. Citation Format: Daniel A. Lichtenstein, Seth M. Steinberg, Steven L. Highfill, Bonnie Yates, Ping Jin, Jianjian Jin, Sandhya Panch, Haneen Shalabi, David F. Stroncek, Terry J. Fry, Nirali N. Shah. Factors predictive of CAR T cell associated hemophagocytic lymphohistiocytosis (HLH) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4231.

Biological characteristics of endometriotic mesenchymal stem cells isolated from ectopic lesions of patients with endometriosis

BackgroundResearch into the pathogenesis of endometriosis (EMs) would substantially promote its effective treatment and early diagnosis. However, the aetiology of EMs is poorly understood and controversial despite the progress in EMs research in the last several decades. Currently, accumulating evidence has shed light on the importance of endometrial stem cells (EnSCs) residing in the basal layer of endometrium in the establishment and progression of endometriotic lesions. Therefore, we aimed to identify the differences between EnSCs isolated from the ectopic lesions of EMs patients (EnSC-EM-EC) and EnSCs isolated from eutopic endometrium of control group (EnSC-Control). We further performed preliminary exploration of the potential signalling pathways involved in the above abnormalities.MethodsEnSC-EM-EC (n = 12) and EnSC-Control (n = 13) were successfully isolated. Then, the proliferative capacity, migratory capacity and angiogenic potential of EnSCs were evaluated by conventional MTT assay, flow cytometry, wound healing assay, transwell assay, tube formation assay and chick embryo chorioallantoic membrane assay respectively. The expression of 11 angiogenesis-associated biological factors and 11 cytokines secreted by EnSCs and 17 adhesion molecules expressed on EnSCs were determined by protein array assays respectively. Differentially expressed genes (DEGs) between EnSC-EM-EC and EnSC-Control were analysed by RNA-sequence.ResultsEnSC-EM-EC exhibited unique biological characteristics, including prolonged mitosis, enhanced migratory capacity and enhanced angiogenic potential. Greater amounts of angiogenic factors (especially VEGF and PDGF) were secreted by EnSC-EM-EC than by EnSC-Control; however, the distinct profiles of cytokines secreted by EnSC-EM-EC and adhesion molecules expressed by EnSC-EM-EC require further investigation. A total of 523 DEGs between EnSC-EM-EC and EnSC-Control were identified and analysed using the KEGG and Gene Ontology databases.ConclusionsOur results not only improve the understanding of EMs but also contribute to the development of EnSC-EM-EC as a tool for EMs drug discovery. These cells could be of great help in exploiting promising therapeutic targets and new biomarkers for EMs treatment and prognosis.

Advanced Cell Mapping Visualizations for Single Cell Functional Proteomics Enabling Patient Stratification.

Highly multiplexed single-cell functional proteomics has emerged as one of the next-generation toolkits for a deeper understanding of functional heterogeneity in cell. Different from the conventional population-based bulk and single-cell RNA-Seq assays, the microchip-based proteomics at the single-cell resolution enables a unique identification of highly polyfunctional cell subsets that co-secrete many proteins from live single cells and most importantly correlate with patient response to a therapy. The 32-plex IsoCode chip technology has defined a polyfunctional strength index (PSI) of pre-infusion anti-CD19 chimeric antigen receptor (CAR)-T products, that is significantly associated with patient response to the CAR-T cell therapy. To complement the clinical relevance of the PSI, a comprehensive visualization toolkit of 3D uniform manifold approximation and projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE) in a proteomic analysis pipeline is developed, providing more advanced analytical algorithms for more intuitive data visualizations. The UMAP and t-SNE of anti-CD19 CAR-T products reveal distinct cytokine profiles between nonresponders and responders and demonstrate a marked upregulation of antitumor-associated cytokine signatures in CAR-T cells from responding patients. Using this powerful while user-friendly analytical tool, the multi-dimensional single-cell data can be dissected from complex immune responses and uncover underlying mechanisms, which can promote correlative biomarker discovery, improved bioprocessing, and personalized treatment development.

Enhancing responsiveness of human jejunal enteroids to host and microbial stimuli.

Enteroids are a physiologically relevant model to examine the human intestine and its functions. Previously, the measurable cytokine response of human intestinal enteroids has been limited following exposure to host or microbial pro-inflammatory stimuli. Modifications to enteroid culture conditions facilitated robust human cytokine responses to pro-inflammatory stimuli. This new human enteroid culture methodology refines the ability to study microbiome:human intestinal epithelium interactions in the laboratory. The intestinal epithelium is the primary interface between the host, the gut microbiome and its external environment. Since the intestinal epithelium contributes to innate immunity as a first line of defence, understanding how the epithelium responds to microbial and host stimuli is an important consideration in promoting homeostasis. Human intestinal enteroids (HIEs) are primary epithelial cell cultures that can provide insights into the biology of the intestinal epithelium and innate immune responses. One potential limitation of using HIEs for innate immune studies is the relative lack of responsiveness to factors that stimulate epithelial cytokine production. We report technical refinements, including removal of extracellular antioxidants, to facilitate enhanced cytokine responses in HIEs. Using this new method, we demonstrate that HIEs have distinct cytokine profiles in response to pro-inflammatory stimuli derived from host and microbial sources. Overall, we found that host-derived cytokines tumour necrosis factor and interleukin-1α stimulated reactive oxygen species and a large repertoire of cytokines. In contrast, microbial lipopolysaccharide, lipoteichoic acid and flagellin stimulated a limited number of cytokines and histamine did not stimulate the release of any cytokines. Importantly, HIE-secreted cytokines were functionally active, as denoted by the ability of human blood-derived neutrophil to migrate towards HIE supernatant containing interleukin-8. These findings establish that the immune responsiveness of HIEs depends on medium composition and stimuli. By refining the experimental culture medium and creating an environment conducive to epithelial cytokine responses by human enteroids, HIEs can facilitate exploration of many experimental questions pertaining to the role of the intestinal epithelium in innate immunity.

Noncoding RNAs modify GATA3-driven transcription, leading to the immune profiles seen in psoriasis and atopic dermatitis

Abstract Atopic dermatitis (AD) and psoriasis are clinical manifestations of immune dysregulation in the skin. Psoriasis is generally classified as a T cell-mediated, IFN-γ dependent disease, while AD is skewed toward interleukin (IL)-4 dependence. Consistent with these classifications are clinical observations that IL-4 improves psoriasis disease score and an IL-4 receptor blocking antibody is approved for treatment of severe AD. However, the immunobiology is far more complex, as IL-13, often produced by IL-4 dependent T cells, can be elevated in psoriasis. Since GATA3 controls both IL-4 and IL-13 expression, we propose that regulation of GATA3 differs between the two diseases, resulting in overlapping yet distinct cytokine profiles. Our meta-analysis of two RNA sequencing profiles for each disease identified a noncoding RNA of unknown function (NRUF) in all four datasets (p&amp;lt;1×10−10). Upon analyzing 48 datasets of immune responses to viral infection in epithelial cells and leukocytes, we found IL-13 and NRUF expression levels are most strongly correlated in lymphocytes. In T cell lines, we found elevated expression of the NRUF noncoding RNA associates with IL-4 and IL-13. However, IL-31, which is elevated in AD independently from the GATA3/IL-4/IL-13 axis, is not correlated to NRUF expression, supporting our hypothesis that NRUF drives disease pathology via the GATA3/IL-4/IL-13-dependent inflammatory response. Using overexpression or silencing approaches, we have characterized the ability of this NRUF to direct cytokine polarization of T cells from healthy control and donors with AD or psoriasis. We propose this NRUF modulates these two cutaneous diseases by regulating GATA3-based IL-4/IL-13 balance in T cells.

Mesenchymal Stem/Stromal Cell Engulfment by Breast Cancer Cells Generates a Hybrid Cancer Cell Population with Dormancy and Chemoresistance

IntroductionDrug‐resistant dormant tumor cells are a major causes of breast cancer relapse and metastasis. It has been reported that in the tumor microenvironment breast cancer cells (BCC) can engulf mesenchymal stem/stromal cells (MSCs) and acquire dormancy. We previously documented the presence of BCC that engulf MSCs in clinical samples of breast cancer metastasis and showed that MSC engulfment leads to a more aggressive breast cancer phenotype, with an upregulated gene signature consisting of MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1. However, the mechanisms of MSC engulfment, and the phenotypic features of hybrid breast cancer cells are still unclear. Here, we tested the hypothesis that MSC engulfment generates hybrid cancer cells exhibit a multinucleated phenotype and increased survival advantage, dormancy, and chemoresistance, and that the genes we identified may mediate MSC engulfment.MethodMSC labeled with DS‐RED and MDA‐MB‐231 labeled with GFP (GFP‐231) were co‐cultured in microfluidic cell pairing devices, and treated with antibodies against MSR1, WNT5A, ELMO1, IL1RL2, ZPLD1 and SIRPB1 or IgG control to quantify the percentage of MSC engulfing on‐chip. The conditioned media was subjected to a cytokine array. Live Imaging Microscopy was used to characterize the hybrid cancer cells in co‐cultures compared the GFP‐231. In Co‐cultures of DS‐RED‐MSC with GFP‐231 stably transduced with shWNT5A, shMSR1 or lentivirus control, and treated with Doxorubicin or untreated, we analyzed the percentage of DS‐RED+/GFP+ hybrid population, Ki‐67 low cells in G1, multinucleated cells (N4+), and the percentage of apoptotic cells using annexin V/PI.ResultsNeutralizing antibodies or shRNAs against WNT5A and MSR1 significantly reduced the percentage of MSC engulfment by MDA‐MB‐231 cells, and changed the cytokine profile in the conditioned medium of co‐cultures. In co‐cultures with DS‐RED‐MSCs, DS‐RED+/GFP+ hybrid cells had a higher percentage of Ki‐67 low in G1 and higher percentage of aneuploidy compared to GFP‐231+ cells, which were reversed by WNT5A and MSR1 shRNA downregulation. Doxorubicin treatment increased the percentage of DS‐RED+/GFP+ hybrids cells and decreased the percentage of apoptosis in the co‐cultures when compared to untreated controls.ConclusionsMSC engulfment by BCCs results in a hybrid multinucleated cell population with increased percentage of aneuploidy than BCC controls and a dormant phenotype characterized by higher of Ki‐67 low in G1 compared to controls. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of pro‐metastatic proteins including thrombosponding‐1, UPAR, and VEGF. Hybrid cancer cells show increased resistance to Doxorubicin compared to control BCCs, which can be rescued by downregulation of WNT5A and MRS1.Support or Funding InformationRole of EZH2 Breast Cancer Progression – R01 to C. Kleer. Role of CCN6 (WISP3) in the progression and metastasis – R01 to C. Kleer‐ AIRC Investigator Grant (IG#18602) to S. Andò‐PRIN2015#2015B7M39T to S. Andò

IL6 Induces an IL22+ CD8+ T-cell Subset with Potent Antitumor Function.

CD8+ T cells can be polarized into several different subsets as defined by the cytokines they produce and the transcription factors that govern their differentiation. Here, we identified the polarizing conditions to induce an IL22-producing CD8+ Tc22 subset, which is dependent on IL6 and the aryl hydrocarbon receptor transcription factor. Further characterization showed that this subset was highly cytolytic and expressed a distinct cytokine profile and transcriptome relative to other subsets. In addition, polarized Tc22 were able to control tumor growth as well as, if not better than, the traditional IFNγ-producing Tc1 subset. Tc22s were also found to infiltrate the tumors of human patients with ovarian cancer, comprising up to approximately 30% of expanded CD8+ tumor-infiltrating lymphocytes (TIL). Importantly, IL22 production in these CD8+ TILs correlated with improved recurrence-free survival. Given the antitumor properties of Tc22 cells, it may be prudent to polarize T cells to the Tc22 lineage when using chimeric antigen receptor (CAR)-T or T-cell receptor (TCR) transduction-based immunotherapies.

Distinct cytokine profiles across trajectories of self-perceived cognitive impairment among early-stage breast cancer survivors

The aim of the current study is to identify distinct cytokine profiles in relation to self-perceived cognitive trajectories. In our study cohort (n = 128), early-stage breast cancer patients were categorized into no impairment reported, acute, delayed, persistent and intermittent cognitive decline respectively. Pro-inflammatory cytokines were elevated compared to baseline; with TNF-α implicated in the acute cognitive trajectory while IL-6 and IL-8 were involved in the persistent cognitive trajectory. Our findings help to further our understanding of cytokine profiles implicated in cancer-related cognitive impairment (CRCI) and support the use of cytokine levels as biomarkers of cognitive decline over time.

HIV-1 Central Nervous System Compartmentalization and Cytokine Interplay in Non-Subtype B HIV-1 Infections in Nigeria and Malawi.

HIV-1 compartmentalization in the central nervous system (CNS) and its contribution to neurological disease have been well documented. Previous studies were conducted among people infected with subtypes B or C where CNS compartmentalization has been observed when comparing viral sequences in the blood to virus in cerebrospinal fluid (CSF). However, little is known about CNS compartmentalization in other HIV-1 subtypes. Using a deep sequencing approach with Primer ID, we conducted a cross-sectional study among Nigerian and Malawian HIV-1 cohorts with or without fungal Cryptococcus infection diagnosed as cryptococcal meningitis (CM) to determine the extent of CSF/CNS compartmentalization with CM. Paired plasma and CSF samples from 45 participants were also analyzed for cytokine/chemokine levels. Viral populations comparing virus in the blood and the CSF ranged from compartmentalized to equilibrated, including minor or partial compartmentalization or clonal amplification of a single viral sequence. The frequency of compartmentalized viral populations in the blood and CSF was similar between the CM- and CM+ participants. We confirmed the potential to see compartmentalization with subtype C infection and have also documented CNS compartmentalization of an HIV-1 subtype G infection. Cytokine profiles indicated a proinflammatory environment, especially within the CSF/CNS. However, sCD163 was suppressed in the CSF in the presence of CM, perhaps due to elevated levels of IL-4, which were also a feature of the cytokine profile, showing a distinct cytokine profile with CM.

Unique Pro-Inflammatory Response of Macrophages during Apoptotic Cancer Cell Clearance.

The clearance of apoptotic cells by macrophages (efferocytosis) is crucial to maintain normal tissue homeostasis; however, efferocytosis of cancer cells frequently results in inflammation and immunosuppression. Recently, we demonstrated that efferocytosis of apoptotic prostate cancer cells by bone marrow-derived macrophages induced a pro-inflammatory response that accelerated metastatic tumor growth in bone. To evaluate the microenvironmental impact of macrophages and their efferocytic function, we compared peritoneal macrophages (P-MΦ) versus bone marrow-derived macrophages (BM-MΦs) using an efferocytosis in vitro model. The capability to engulf apoptotic prostate cells was similar in BM-MΦs and P-MΦs. Ex vivo analysis of BM-MΦs showed an M2-like phenotype compared with a predominantly M1-like phenotype in P-MΦs. A distinct gene and protein expression profile of pro-inflammatory cytokines was found in BM-MΦs as compared with P-MΦs engulfing apoptotic prostate cancer cells. Importantly, the reprogramming of BM-MΦs toward an M1-like phenotype mitigated their inflammatory cytokine expression profile. In conclusion, BM-MΦs and P-MΦs are both capable of efferocytosing apoptotic prostate cancer cells; however, BM-MΦs exert increased inflammatory cytokine expression that is dependent upon the M2 polarization stage of macrophages. These findings suggest that bone marrow macrophage efferocytosis of apoptotic cancer cells maintains a unique pro-inflammatory microenvironment that may support a fertile niche for cancer growth. Finally, bone marrow macrophage reprogramming towards M1-type by interferon-γ (IFN-γ) induced a significant reduction in the efferocytosis-mediated pro-inflammatory signature.

The specific ex vivo released cytokine profile is associated with ischemic stroke outcome and improves its prediction

BackgroundInflammation is associated with poor outcome after stroke. A relationship between ex vivo cytokine synthesis and stroke outcome remains unclear. We explored an association between ex vivo cytokine release, circulating interleukin (IL)-6 as a marker of systemic inflammation, and stroke prognosis. We assessed the utility of ex vivo synthesized cytokines for predicting stroke outcome.MethodsWe collected blood from 248 ischemic stroke patients and stimulated it ex vivo with lipopolysaccharide. We measured concentration of synthesized cytokines (TNFα, IP-10, IL-1β, IL-6, IL-8, IL-10, and IL-12) and plasma IL-6. We assessed functional outcome 3 months after stroke using the modified Rankin Scale. To assess the prognostic ability of cytokines, we applied multivariate logistic regression, cluster analysis, and construction of multimarker score.ResultsDecreased release of IP-10, TNFα, IL-1β, and IL-12; increased release of IL-10 and IL-8; and higher plasma IL-6 level were associated with poor outcome. Cluster analysis identified three groups of patients with distinct cytokine profiles. The group with the worst outcome demonstrated high synthesis of IL-10, IL-8, IL-1β, and IL-6 and low synthesis of IL-12, IP-10, and TNFα accompanied by high circulating IL-6 level. The group with the best prognosis showed high synthesis of TNFα, IP-10, IL-12, IL-1β, and IL-6; low synthesis of IL-10 and IL-8; and low plasma IL-6. Patients with intermediate outcome had low synthesis of all cytokines accompanied by low circulating IL-6. We constructed a multimarker score composed of ex vivo released IL-12, IL-10, TNFα, and plasma IL-6. Addition of this score to clinical variables led to significant increase in c-statistic (0.81 vs 0.73, p = 0.02) and net reclassification improvement.ConclusionThe decreased ex vivo release of pro-inflammatory cytokines and increased release of IL-10 and IL-8 are related to poor outcome after stroke. Cytokine-based multimarker score adds prognostic value to clinical model for predicting stroke outcome.

Classifying dementia progression using microbial profiling of saliva.

There is increasing evidence linking periodontal infections to Alzheimer's disease (AD). Saliva sampling can reveal information about the host and pathogen interactions that can inform about physiological and pathological brain states. A cross-sectional cohort of age-matched participants (78) was segmented according to their chemosensory (University of Pennsylvania Smell Identification Test; UPSIT) and cognitive scores (Mini-Mental State Exam; MMSE and clinical dementia rating; CDR). Mid-morning saliva was sampled from each participant and processed for microbiome composition and cytokine analysis. Linear discriminant analysis (LDA) was used to unravel specific changes in microbial and immunological signatures and logistic regression analysis (LRA) was employed to identify taxa that varied in abundance among patient groups. Using olfaction we distinguish in the cognitively normal population a segment with high chemosensory scores (CNh, 27) and another segment with chemosensory scores (CNr, 16) as low as mild cognitive impairment (MCI, 21) but higher than the AD group (17). We could identify stage-specific microbial signatures changes but no clearly distinct cytokine profiles. Periodontal pathogen species as Filifactor villosus decline with the increasing severity of AD, whereas opportunistic oral bacteria such as Leptotrichia wadei show a significant enrichment in MCI. The salivary microbiome indicates stage-dependent changes in oral bacteria favoring opportunistic species at the expense of periodontal bacteria, whereas the inflammatory profiles remain mainly unchanged in the sampled population.

Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients

ABSTRACT Circulating in China and 158 other countries and areas, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public health. However, efforts to identify effectively supportive therapeutic drugs and treatments has been hampered by our limited understanding of host immune response for this fatal disease. To characterize the transcriptional signatures of host inflammatory response to SARS-CoV-2 (HCoV-19) infection, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients. Our results reveal distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release such as CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B. Furthermore, SARS-CoV-2 induced activation of apoptosis and P53 signalling pathway in lymphocytes may be the cause of patients’ lymphopenia. The transcriptome dataset of COVID-19 patients would be a valuable resource for clinical guidance on anti-inflammatory medication and understanding the molecular mechansims of host response.

Isolation, Culture, and Induction of Plasticity in ILC2s.

Innate lymphoid cells (ILCs) are lymphocytes with critical roles in homeostasis, inflammation, and immunity to pathogens. ILCs are rare relative to other immune cell populations and are primarily defined by lack of expression of markers associated with other immune cell lineages and are predominantly found in mucosal tissues like the gut, lung and skin. They are classified into distinct subsets, ILC1, ILC2, and ILC3, which mirror subsets of CD4+ helper T cells. ILC subsets have distinct cytokine and transcription factor profiles which align with their biological functions, although recently it has emerged that ILC subsets are not phenotypically fixed and exhibit considerable heterogeneity and plasticity in different contexts. Here, we describe protocols for the maintenance, expansion, and induction of plasticity in mouse and human ILC2s. The resulting cells can be used for molecular interrogation of ILC function and biology, both in vivo and in vitro.