Absence Of IL-4 Research Articles

IL-4 absence triggers distinct pathways in apical periodontitis development

Dental pulp is a specialized tissue able to respond to infectious processes. Nevertheless, infection progress and root canal colonization trigger an immune-inflammatory response in tooth-surrounding tissues, leading to apical periodontitis and bone tissue destruction, further contributing to tooth loss. In order to shed some light on the effects of IL-4 on periradicular pathology development modulation, microtomographic, histological and proteomic analyses were performed using 60 mice, 30 wild type and 30 IL-4−/−. For that, 5 animals were used for microtomographic and histological analysis, and another 5 for proteomic analysis for 0, 7 and 21 days with/without pulp exposure. The periapical lesions were established in WT and IL-4−/− mice without statistical differences in their volume, and the value of p < 0.05 was adopted as significant in microtomographic and histological analyses. Regarding histological analysis, IL-4−/− mice show aggravation of pulp inflammation compared to WT. By using proteomic analysis, we have identified 32 proteins with increased abundance and 218 proteins with decreased abundance in WT animals after 21 days of pulp exposure, compared to IL-4−/− animals. However, IL-4−/− mice demonstrated faster development of apical periodontitis. These animals developed a compensatory mechanism to overcome IL-4 absence, putatively based on the identification of upregulated proteins related to immune system signaling pathways.Significance: IL-4 might play a protective role in diseases involving bone destruction and its activity may contribute to host protection, mainly due to its antiosteoclastogenic action.

Dynamics of cytokines in patients with enteral insufficiency in advanced peritonitis

In this work, changes in the cytokine profile in patients with common forms of peritonitis with enteral insufficiency were studied. The obtained results were compared in patients who survived after surgery and programmed peritoneal sanation and patients who died in the early postoperative period. The aim of the study was to investigate the dynamics of cytokine profile of patients with common forms of peritonitis complicated by enteral insufficiency in order to determine their prognostic value. Material and methods. The object of the study was the blood of 23 patients with common forms of peritonitis and 17 healthy volunteers. To assess the condition of patients performed a dynamic determination in a comprehensive examination of laboratory markers of endogenous toxemia and systemic inflammatory response syndrome: immunological changes in patients by examining blood cells expressing clusters of CD11a+, CD162+, CD95+, CD16+, and molecules of the main histocompatibility complex HLA-DR+ and the level of interleukins IL-2, IL-4, IL-6. Results. During the study in patients with widespread peritonitis complicated by enteral insufficiency, at the time of hospitalization, there was a six-fold decrease in blood levels of CD11a+ cells and the content of CD162+ and CD16+ cells, respectively, 2,8 and 2 times, compared with healthy people. It has been shown that a single remediation programmed relaparotomy within two weeks effectively corrects these changes except for expression on CD16+ cells, which indicates a low effect of this type of surgery on the elimination of immune complexes. It was found that the concentration in plasma IL-2 increased 5 times and was 27,1% higher than that in almost healthy individuals. At the same time, the level of IL-4 in the blood decreased sharply – 4 times. Plasma IL-6 content also reduced. One week after relaparotomy, the expression of HLA-DR + molecules on immunocompetent cells increased again and was 1,8 times higher than the control parameters. In addition, the content of cytokines in the blood plasma increased sharply: the level of IL-2 exceeded the control by 2,4 times, IL-4 by 4 times, and IL-6 by 2 times. Conclusions. 1. Progressive and noticeable decrease in IL-2 content occurs in patients with widespread peritonitis complicated by enteral insufficiency in the complete absence of IL-4 in the blood on the background of a permanent and significant increase in plasma concentrations of IL-6. 2. Under conditions of suppression of the immune response, due to intoxication of the patient's body, the mechanism of endogenous inhibition of the immune response "IL-6 – corticoliberin – corticotropin – corticosteroids" is additionally realized.

Research Highlights

Longevity and Replenishment of Human Liver-resident Memory T Cells and Mononuclear Phagocytes Pallett LJ, Burton AR, Amin OE, et al. J Exp Med. 2020;217(9):e20200050. There is an increasing interest in tissue resident leukocytes and their role in mediating rejection and tissue regeneration after transplantation.1 However, not a great deal is known about graft-resident populations in the liver after transplantation.2 Pallett et al3 investigated the composition of liver-resident immune cells in HLA-mismatched donor–recipient pairs after transplantation. They found that the majority of cells were recipient-derived cells recruited from the periphery but that a donor-derived population persisted in the graft, even up to 10 years after transplantation. Interestingly, this appeared to be unique to the liver, as no such chimerism was detected in the peripheral blood. Additionally, certain populations including polymorphonuclear cells, NK, T, and NKT cells persisted more effectively than other cell types, such as dendritic cells. The infiltrating cells acquired tissue-resident and liver-resident features and even replicated the ratios of cell types normally found in the liver, such as the higher CD8:CD4 ratio that is normally found in the periphery. This suggests that the liver microenvironment can recruit cells and alter their phenotype to fit liver-resident requirements, regardless of HLA provenance. The infiltrating cells acquired phenotypic markers including the expression of Kupffer/macrophage cell marker CD68 but still were distinguishable from embryonically seeded donor cells in the expression of a few markers, such as HMOX1, CD206, and CD163, indicating that the phenotype induced is not identical to native tissue-resident cells. This phenotypic malleability has implications in cellular therapy, if the graft is capable of controlling which cell types it recruits and is capable of then morphing their phenotype according to its homeostatic composition. The presence of a long-lived donor-derived resident population is also of interest for localized chimerism within the graft, which may help facilitate tolerance.4 This is particularly relevant as donor T cells persisted, but dendritic cells did not. Whether this long-lived chimerism is restricted to the liver or can be achieved in other organs is a matter of great relevance for adoptive cellular therapies. Sympathetic Nervous Tone Limits the Development of Myeloid-derived Suppressor Cells Nevin JT, Moussa M, Corwin WL, et al. Sci Immunol. 2020;5(51):eaay9368. The immune system can respond to a huge variety of physiological cues. Sympathetic nervous system (SNS) release of norepinephrine directly signals to various immune cells via adrenergic receptors (ARs) expressed on their surface, known to modify their function.1 In the study from Nevin et al,2 the authors investigated the impact of ablation of SNS signaling on myeloid cells in mice. Sympathetic signaling was targeted using neurotoxin 6-hydroxydopamine, which is selectively taken up by sympathetic terminals and results in their degradation. Sympathectomized mice had increased polymorphonuclear (PMN) and monocytic cell populations with phenotypes consistent with polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) and mononuclear MDSCs (M-MDSCs). They found that the CD11b+ compartment from sympathectomized mice was more suppressive than that of control mice, specifically the CD11b+Ly6CintLy6G+ PMN-MDSC subpopulation. Single-cell RNA sequencing revealed a shift in the PMN population in SNS-ablated mice, with an enrichment of immature cells and a decrease in cells expressing the mature neutrophil marker CD101, as well as an increase in proliferation. Sympathectomized mice also had an increase in regulatory T cell (Treg) populations in a Gr1-dependent manner. The mechanism behind this was further elucidated with the use of α-AR and ß-AR agonists and antagonists, which revealed that ß-AR agonism and α-AR antagonism both reproduced the effects of SNS ablation and induced MDSC proliferation and impaired maturity. As α-ARs have higher affinity for norepinephrine than ß-ARs, the researchers suggest that tonal SNS signaling preferentially affects α-ARs and that SNS ablation therefore reduces this to induce MDSC development. Additionally, they suggest that s100a8-s100a9 heterodimers secreted by MDSCs may be responsible for the observed Treg induction, as this heterodimer has been shown to signal via CD69 to induce STAT5 phosphorylation. Indeed, iTreg induced in the presence of s100a8-s100a9 heterodimers showed increased Foxp3 expression and proliferation even in the absence of IL-2, suggesting that this dimer can compensate via alternative STAT5 phosphorylation. Naturally suppressive cell populations such as Tregs and MDSCs are of significant interest to the field of transplantation for their ability to therapeutically control alloresponses.3,4 Understanding the various physiological changes that modify leukocyte behavior after transplantation is critical. This study sheds some light on the physiological mechanisms that control these leukocyte populations and has implications for transplantation where transplanted organs are denervated or the settings of organ failure and surgery in which there is autonomic system activation.

The Central Role of MAPK-ERK Signaling in IL7-Dependent and IL7-Independent Steroid Resistance Reveals a Broad Application of MEK-Inhibitors Compared to JAK1/2-Inhibition in T-ALL

Introduction Poor prednisolone response in the induction phase of treatment is a risk-stratification marker in the current Dutch DCOG ALL-11 treatment protocol. Physiological or mutational activation of the IL7-receptor (IL7R) provokes steroid resistance in T-ALL. Moreover, relapsed T-ALL patients that harbor IL7R or RAS mutations are considered as an ultra-high-risk group with extremely poor outcome. Aim We studied the contribution of two key IL7R downstream pathways (STAT5B and MAPK-ERK respectively) to steroid resistance in order to optimize future treatment regimens. Results To date, IL7-dependent steroid resistance has been proposed to depend on IL7-induced JAK-STAT signaling and subsequent BCL2 upregulation. Here, we demonstrate that the transforming STATBN642H mutation does not impair steroid sensitivity despite a paradoxical upregulation of BCL2 and BCLXL following steroid treatment. We therefore hypothesized that other pathways contribute to IL7-dependent steroid resistance in T-ALL. By studying T-ALL PDX samples, we observed that physiological IL7-signaling can activate the MAPK-ERK pathway in IL7-dependent and IL7-independent steroid resistant PDX cells. Similar to mutational-driven MAPK-ERK signaling (e.g. by activating IL7R, JAK1 or NRAS mutations), IL7-induced MAPK-ERK signaling leads to the inactivation of the pro-apoptotic protein BIM by inhibitory phosphorylation of both BIM-EL and BIM-L isoforms. By mass spectrometry, we observe that phosphorylated BIM specifically decreases its physical interaction with Aurora kinase A and Bcl-2 family members BMF, BCL2, BCLXL and MCL1, which alters the apoptotic threshold and effectuates steroid resistance. The JAK1/2-inhibitor ruxolitinib and MEK-inhibitor selumetinib are considered for future basket-treatment protocols in IL7R/JAK-STAT-activated or MAPK-ERK-activated leukemias respectively. We demonstrate that both ruxolitinib and selumetinib prevent IL7-induced MAPK-ERK signaling in T-ALL PDX cells. Moreover, ruxolitinib and selumetinib abolish BIM phosphorylation in mutant JAK1-overexpressing SUPT-1 cells, which restores the binding of BIM to BCL2, BCLXL and MCL1. Only selumetinib restores the function of BIM in wild type or mutant NRAS overexpressing SUPT-1 cells, indicating that ruxolitinib only blocks MAPK-ERK signaling in the context of 'upstream' (e.g. mutant IL7R/JAK1 or IL7-induced) pathway activation. Surprisingly, none of our 46 PDX samples respond to ruxolitinib treatment in the absence of IL7, while the majority of these samples demonstrate a robust response towards MEK-inhibition. IL7-dependent steroid resistant PDX samples do respond to ruxolitinib treatment in the presence of IL7, while IL7 exposed IL7-independent steroid resistant PDX samples remain unresponsive to ruxolitinib. We therefore conclude that the therapeutic effect of ruxolitinib is dependent on an IL7-protective effect, limiting its therapeutic application to a selective subset of T-ALL patients. When we combine prednisolone treatment with selumetinib or ruxolitinib, we observe that ruxolitinib only synergizes with prednisolone in IL7-dependent steroid resistant PDX samples in the presence of IL7. Importantly, combination treatment with prednisolone and selumetinib acts highly synergistic in IL7-dependent and IL7-independent steroid resistant samples in the presence and absence of IL7. Conclusion and clinical relevance In addition to described JAK-STAT and PI3K-AKT signaling, our study demonstrates that IL7-induced signaling activates the MAPK-ERK signaling pathway, which actively contributes to IL7-dependent steroid resistance. We demonstrate the central importance of MAPK-ERK signaling in T-ALL, whereas all T-ALL PDX samples tested highly benefit from combined prednisolone and selumetinib treatment. Ruxolitinib only acts synergistically with steroids in the context of IL7-dependent steroid resistance. Moreover, our data proposes that this synergistic effect may (in part) depend on the anti-MAPK-ERK effect downstream of JAK1/2-inhibition. Combined, our study strongly supports the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507), and contributes to the optimization and stratification of newly designed T-ALL treatment regimens. Disclosures Bourquin: Servier: Other: Travel Support. Vormoor:AstraZeneca: Research Funding.

Inhibitory role of interleukin-10 in the cutaneous reverse Arthus reaction.

The formation and deposition of immune complexes (IC) containing immunoglobulin (Ig)G antibodies induces an acute inflammatory response with tissue injury. One of the experimental models of IC-related vasculitis is the cutaneous reverse passive Arthus reaction, in which IgG antibodies are injected i.d., followed immediately by the i.v. application of the corresponding antigen. This reaction is characterized by edema, hemorrhage and neutrophil infiltration. To assess the role of the anti-inflammatory cytokine interleukin (IL)-10 in IC-related vasculitis, we investigated the cutaneous Arthus reaction using IL-10 knockout (IL-10KO) mice. Edema, which was quantified macroscopically by measuring the vascular leakage of Evans blue dye at 4h after IC challenge, was significantly increased in IL-10KO mice compared with wild-type mice. In addition, hemorrhage, which was assessed by the average diameter of purpuric spots at 8h after IC challenge, was enhanced significantly in IL-10KO mice compared with wild-type mice. Histological examination showed that the number of extravascular neutrophils was significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8h after IC challenge. Analysis of pro-inflammatory cytokine mRNA expression showed that IL-6 mRNA levels were significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8h after IC challenge. These results showed that IC-induced inflammation and vascular damage were significantly enhanced in the absence of IL-10. Thus, IL-10 may limit tissue disruption by suppressing the excessive infiltration of neutrophils and cytokine expression in a mouse model of type III vasculitis.

CYTOKINE REGULATION OF THE IMMUNE RESPONSE IN PATIENTS WITH ACUTE NECROTIC PANCREATITIS COMPLICATED BY MULTI-ORGANIC FAILURE

Aim: To study the cytokine regulation of the immune response in patients with acute necrotic pancreatitis (ANP) complicated by multiple organ failure (MON).&#x0D; Material and methods: A prospective cohort study of 22 patients with ANP complicated by MON who were treated in the clinic during 2014-2020 has been performed. We studied changes in blood cells expressing clusters CDIIa +, CD162 +, CD95 +, CD16 +, HLA-DR + molecules, levels of interleukins IL-2, IL-4, IL-6. The efficacy of treatment was determined by the duration of the organ failure, the level of postoperative complications and mortality.&#x0D; Results: Low expression of CD11a + and CD162 + on immunocompetent cells together with two time reduction of the content of CD95 + cells was observed in patients with ANP complicated by MON, which caused a decrease in the concentration of IL-6 at the same time with a slight increase in IL-4. A permanent reduction of the content of CD11a+-, CD162+- and CD95+-leukocytes simultaneously with a drop of the concentration of IL-4 and an excessive increase in the levels of IL-2 and IL-6 was associated with a negative course of the disease.&#x0D; Conclusions: The imbalance of cytokine regulation of the immune response develops in patients with ANP, complicated by MON. Progressive and excessive increase of plasma concentrations of IL-2 and IL-6 occurs as well as complete absence of IL-4 occurs in patients with an adverse course of the disease.

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

Eosinophil-Mediated Immune Control of Adult Filarial Nematode Infection Can Proceed in the Absence of IL-4 Receptor Signaling.

Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R-dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Rα-deficient (IL-4Rα-/-) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Rα-/- mice. By treating IL-4Rα-/- mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Rα-/- mice, residual eosinophilia was ablated, and susceptibility to chronic adult Brugia malayi infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL-13-dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Rα signaling and defined that IL-4Rα-/-/IL-5-/- double-knockout mice displayed significant eosinophil deficiency compared with IL-4Rα-/- mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R-dependent or IL-4R-independent/CCR3/IL-5-dependent immunity influenced B. malayi microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R-independent/IL-5- and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency.

Protective role of Galectin-7 for skin barrier impairment in atopic dermatitis.

BackgroundAtopic dermatitis (AD) patients have a barrier disorder in association with Th2 dominant skin inflammation. Galectin‐7 (Gal‐7), a soluble unglycosylated lectin, is highly expressed in the stratum corneum of AD patients. However, the biological significance of increased Gal‐7 expression in AD skin lesions remains unclear.ObjectiveWe aimed to investigate the production mechanism and functional role of Gal‐7 in AD patients and IL‐4/IL‐13–stimulated epidermal keratinocytes.MethodsWe assessed the Gal‐7 expression levels in skin lesions and sera from AD patients. Gal‐7 levels were also measured in monolayered normal human epidermal keratinocytes (NHEKs) and 3‐dimensional (3D)–reconstructed epidermis in the presence or absence of IL‐4/IL‐13 with or without Stat3, Stat6 or Gal‐7 gene silencing.ResultsGal‐7 was highly expressed in the stratum corneum or intercellular space of AD lesional epidermis as assessed by the stratum corneum proteome analysis and immunohistochemistry. A positive correlation was noted between serum Gal‐7 level and transepidermal water loss in patients with AD. These clinical findings were corroborated by our in vitro data, which showed that IL‐4/IL‐13 facilitated the extracellular release of endogenous Gal‐7 in both monolayered NHEKs and 3D‐reconstructed epidermis. This machinery was caused by IL‐4/IL‐13–induced cell damage and inhibited by knockdown of Stat6 but not Stat3 in NHEKs. Moreover, we performed Gal‐7 knockdown experiment on 3D‐reconstructed epidermis and the result suggested that endogenous Gal‐7 serves as a protector from IL‐4/IL‐13–induced disruption of cell‐to‐cell adhesion and/or cell‐to‐extracellular matrix adhesion.Conclusion and Clinical RelevanceOur study unveils the characteristic of Gal‐7 and its possible role as an alarmin that reflects the IL‐4/IL‐13–induced skin barrier impairment in AD.

Pharmacological inhibition of RORC2 enhances human Th17-Treg stability and function.

Inflammatory bowel diseases (IBD) are chronic conditions that result from uncontrolled intestinal inflammation. Pathogenic Th17 cells, characterized by production of IL-17A in the absence of IL-10, are thought to contribute to this inflammation, but in humans, antibody-mediated blockade of IL-17A is an ineffective IBD therapy whereas IL-23 blockade is effective. Here, we investigated the effects of pharmacological inhibition of RORC2, the Th17 cell lineage-defining transcription factor, on in vivo-differentiated human Th17 cells and Th17-like Tregs (Th17-Tregs). BMS-336, a small molecule RORC2 inverse agonist, inhibited expression of RORC2-regulated genes in peripheral Th17 cells (CD4+ CD25- CD127+ CXCR3- CCR4+ CCR6+ ) in a dose-dependent manner, with similar inhibitory effects on laminar propria mononuclear cells from IBD and non-IBD subjects. Exposure of peripheral Th17-Tregs (CD4+ CD25hi CD127lo CXCR3- CCR4+ CCR6+ ) to BMS-336 also inhibited IL-17A production and prevented inflammatory cytokine-induced destabilization, as evidenced by preserved FOXP3 expression and epigenetic status of the Treg-specific demethylation region. In parallel, RORC2 inhibition increased the production of IL-10 in Th17-Tregs, resulting in enhanced suppression of inflammatory cytokines from myeloid cells. Thus, via its ability to simultaneously inhibit Th17 cells and enhance the stability and function of Th17-Tregs, pharmacological inhibition of RORC2 is a promising approach to suppress inflammation and promote immune regulation in IBD.

WNT7A Expression is Downregulated in T Lymphocytes after T-Cell Receptor Activation Due to Histone Modifications and in T-ALL by DNA Methylation.

WNT signaling pathway regulates several processes involved in the homeostasis of normal cells. Its dysregulation is associated with pathological outcomes like cancer. We previously demonstrated that downregulation of WNT7A correlates with higher proliferation rates in acute lymphoblastic leukemia. However, the regulation of this gene in pathological and normal conditions remains unexplored. In this work, we aimed to analyze the transcriptional regulation of WNT7A in leukemic cells and in normal T lymphocytes after a proliferative stimulus. WNT7A expression was measured in blood cells and in T lymphocytes after phytohemagglutinin-L (PHA-L) treatment or T-cell receptor (TCR) activation by qPCR and Western blot. Promoter methylation was assessed using methylation-sensitive restriction enzymes, and histone modifications were determined by chromatin immunoprecipitation and qPCR. In T-cell acute lymphoblastic leukemia (T-ALL), WNT7A expression is silenced through DNA methylation of CpG island in the promoter region. In normal peripheral blood cells, WNT7A is mainly expressed by monocytes and T lymphocytes. TCR activation induces the downregulation of WNT7A in normal T lymphocytes by changes in histone methylation marks (H3K4me2/3) and histone deacetylases. A proliferative stimulus mediated by IL-2 keeps WNT7A expression at low levels but in the absence of IL-2, the expression of this gene tends to be restored. Furthermore, after TCR activation and WNT7A downregulation, target genes associated with the WNT canonical pathway were upregulated indicating an independent activity of WNT7A from the WNT canonical pathway. WNT7A expression is silenced by long-term DNA methylation in T-ALL-derived cells and downregulated by histone modifications after TCR activation in normal T lymphocytes.

Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times from 0 to 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.

IL-4 synergizes with low-dose IL-2 to restore systemic lupus erythematosus B cells at the resting naive status

Abstract We have recently identified that down-regulation of interleukin-4 receptor (IL-4R) is a novel hallmark of B-cell dysregulation in SLE patients. Single cell transcriptomics analysis revealed that elevation of IL4R in pre-switched B cells is the opposing signature of increased type I interferon stimulated genes (ISGs) and double negative B cells (TBX21, ITGAX, FCRL3, FCRL5, and ZEB2). Flow cytometry analysis further confirmed that down-regulation IL-4R and upregulation of intracellular IFN-β correlated with the loss of CD21+IgD+CD27− resting naïve (rNVA) B cells and accumulation of CD21−IgD+CD27− activated naïve (aNAV) and double negative CD21−IgD+CD27− B cells in SLE patients (n=47). Interestingly, exposure of SLE B cells to IL-4 prior to treatment with low-dose IL-2 (0.2 ng/mL) blocks the anti-Ig, IL-21, IFN-gamma, TLR7 and BAFF-stimulated development of T-bet+CD11c+IgD−CD27− DN2 B cells and CD27+CD38+ plasmablasts (PBs) and preserves B cells in the rNAV state. Such effect was diminished in the absence of IL-4 or when low-dose of IL-2 was substituted with high-dose of IL-2 (20 ng/mL). Stimulation of B cells with IL-4 promoted the induction of CD25 (IL-2RA) on B cells. Together, our results suggest a mechanism of action underlying the synergism between IL-4 and low-dose IL-2. Specifically, the IL-4R signaling pathways upregulate CD25 on B cells, thereby promoting formation of the high affinity IL-2R complexes required for low-dose IL-2 signaling that induces a regulatory signal that restores SLE B cells to the rNAV stage. Our results suggest that the process of development of autoreactive B cells in SLE can be directly measured in a clinical lab, and can be directly abrogated using cytokine therapy available for clinical use.

Interleukin-10 and non-classical monocytes as biomarkers of ischemic cardiomyopathy

Background. Chronic heart failure frequently occurs against the background of ischemic heart disease (IHD), and has also been associated with the diagnosis of ischemic cardiomyopathy (ICMP). While there are significant commonalities with respect to underlying mechanisms, it would be most important to identify differential diagnostic markers to facilitate diagnosis and verification at the early stages of disease.Aim. The intent of this study was to perform a quantitative assessment of non-classical monocytes in patients diagnosed with IHD with or without ICMP and to identify any relationships between non-classical monocytes and plasma concentrations of proinflammatory (interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines.Methods. We examined 44 patients diagnosed with IHD, aged 49 - 63 years, with class II-III heart failure as per the New York Heart Association criteria. Of this group, 18 were also diagnosed with ICMP. The comparison group included 14 age-matched healthy controls. Percentages of non-classical (CD14+CD16+) monocytes in peripheral blood were determined by flow cytometry and the concentrations of IL-1β, IL-4, IL-6, IL-10, IL-13 and TNF-α were measured by enzyme-linked immunosorbent assay.Results. Amongst the patients diagnosed with ICMP, the concentration of IL-10 in blood plasma was higher than in those without this diagnosis, detected at 30.05 pg/ml (range 24.75 - 33.50 pg/ml, р=0.0412); the percentage of non-classical monocytes was lower, at 5.05% (range 4.08 - 6.58%, р=0.0094). Amongst patients diagnosed with IHD without ICMP, these parameters were within normal limits. The plasma concentrations of IL-1β, IL-6 and IL-13 in both groups were comparable to values obtained from healthy donors and IL-4 was undetectable throughout. Amongst patients diagnosed with ICMP, plasma TNF-α was detected at elevated levels that were comparable to those determined for patients diagnosed with IHD without ICMP.Conclusion. Imbalance of cytokines in blood in patients with coronary artery disease is characterized by excess TNF-α, absence of IL-4, and normal levels of IL-1β, IL-6 and IL-13, regardless of the type of ischemic myocardial injury. In patients with ICMP, we detected an overall decrease in the fraction of non-classical monocytes in association with an increase in plasma IL-10; these features were not detected in patients diagnosed with IHD without ICMP. As such, these biological responses may be used for timely diagnosis of ICMP. Received 27 May 2019. Revised 7 October 2019. Accepted 30 October 2019. Funding: The work is supported by grants of the Russian Foundation for Basic Research No. 18-015-00160\19 and the President of the Russian Federation No. НШ-2690.2018.7 and No. МД-2788.2019.7. Conflict of interest: Authors declare no conflict of interest. Author contributionsConception and study design: V.M. Shipulin, S.P. ChumakovaData collection and analysis: V.M. Shipulin, S.P. Chumakova, O.I. Urazova, M.V. Vins, V.V. NovickiyLiterature review: S.P. Chumakova, D.A. Pogonchenkova, A.S. PryakhinDrafting the article: S.P. Chumakova, D.A. Pogonchenkova, O.I. UrazovaCritical revision of the article: V.M. Shipulin, V.V. NovickiyStatistical analysis: S.P. ChumakovaFinal approval of the version to be published: V.M. Shipulin, S.P. Chumakova, D.A. Pogonchenkova, O.I. Urazova, M.V. Vins, A.S. Pryakhin, V.V. Novickiy

IL-4 enhances interferon production by virus-infected human mast cells

IL-4 enhances interferon production by virus-infected human mast cells

Abstract P6-03-09: Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells

Abstract Background: CDK4/6 inhibitors combined with endocrine therapy (ET) are mainstay to treat metastatic estrogen receptor (ER) positive patients. Yet, almost 60% develop resistance to CDK4/6 inhibition within 2 years of initial treatment. An ongoing clinical challenge has thus been identifying biomarkers of response to predict patients that will either respond or not respond to palbociclib. Further, there is an unmet need to identify actionable targets for patients that have progressed on CDK4/6 blockade regimens. Currently, the only biomarker being used to identify patients for anti-CDK4/6 therapy is estrogen receptor (ER) by IHC. The goal of our study was thus to identify the therapeutic vulnerabilities of CDK4/6 inhibitor resistance cells and identify key markers that can longitudinally correlate with development of resistance. Methods: We have developed MCF7 and T47D resistant cells by treating them with increasing doses (1.2, 2.4, 3.6, and 4.8μΜ) of palbociclib over a 6-month period. At each dose, resistant cells were harvested to observe dose dependent changes that occur as the cells acquire resistance to palbociclib. At endpoint i.e. 4.8μM, multi-omics approach was used to compare sensitive vs. resistant cells. Results: Our multi-omics data indicates that palbociclib resistant cells show i) Upregulation of the STAT3-IL6 pathway ii) Loss of ERα and iii) Loss RB protein levels. Primarily, resistant cells adapt to palbociclib treatment by a dose dependent upregulation of IL6. Further, we observe that induction of IL-6 is an early event which also correlates with senescence-associated secretory phenotype (SASP) signature in resistant cells, predicted to be induced with CDK4/6 inhibition. Importantly, time-course studies using 1μM palbociclib indicates that IL-6 is needed in overcome SASP and promote proliferation in cells adapting and eventually developing resistance to CDK4/6 inhibition. Lastly, we subjected MCF7 and T47D parental cells, which are sensitive to endocrine therapy and palbociclib, with exogenous IL-6 and IL-6 receptor (IL-6R). Treatment with IL-6/IL-6R lead to the downregulation of ER and RB protein levels. IL-6 treated parental cells are 5-fold more resistant to tamoxifen and 7-fold more resistant to fulvestrant compared to parental cells not treated with IL-6. Additionally, dose response studies with palbociclib showed parental cells treated with IL-6 are 5-fold more resistant to palbociclib treatment compared to the cells cultured in the absence of IL-6. Conclusions: Collectively, our data suggests that resistance to palbociclib results in a cascade of events initiating with induction of senescence, leading to sustained upregulation of IL-6 which leads to the promotion of an aggressive tumor growth, accompanied by resistance to endocrine therapy. Our ongoing studies are geared towards delineating the role of each step of CDK4/6 resistance and determining if IL-6 is required to maintain CDK4/6 inhibition resistance. Our long-term goal is to correlate provide the pre-clinical rationale for using IL-6 as a predictive biomarker in patients receiving CDK4/6 inhibitor based therapies. Citation Format: Nicole M Kettner, Tuyen Bui, Xian Chen, Kelly K Hunt, Debu Tripathy, Khandan Keyomarsi. Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-09.

IL-10 Impairs Local Immune Response in Lung Granulomas and Lymph Nodes during Early Mycobacterium tuberculosis Infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be a major global health problem. Lung granulomas are organized structures of host immune cells that function to contain the bacteria. Cytokine expression is a critical component of the protective immune response, but inappropriate cytokine expression can exacerbate TB. Although the importance of proinflammatory cytokines in controlling M. tuberculosis infection has been established, the effects of anti-inflammatory cytokines, such as IL-10, in TB are less well understood. To investigate the role of IL-10, we used an Ab to neutralize IL-10 in cynomolgus macaques during M. tuberculosis infection. Anti-IL-10-treated nonhuman primates had similar overall disease outcomes compared with untreated control nonhuman primates, but there were immunological changes in granulomas and lymph nodes from anti-IL-10-treated animals. There was less thoracic inflammation and increased cytokine production in lung granulomas and lymph nodes from IL-10-neutralized animals at 3-4 wk postinfection compared with control animals. At 8 wk postinfection, lung granulomas from IL-10-neutralized animals had reduced cytokine production but increased fibrosis relative to control animals. Although these immunological changes did not affect the overall disease burden during the first 8 wk of infection, we paired computational modeling to explore late infection dynamics. Our findings support that early changes occurring in the absence of IL-10 may lead to better bacterial control later during infection. These unique datasets provide insight into the contribution of IL-10 to the immunological balance necessary for granulomas to control bacterial burden and disease pathology in M. tuberculosis infection.

188: Absence of IL-6 does not decrease pulmonary inflammation in two models of bronchopulmonary dysplasia (BPD)

BPD is the result of chronic pulmonary inflammation and abnormal alveolar remodeling. IL-6 is an inflammatory mediator which has been implicated in this process. Two models which produce BPD in mice have been previously reported. We sought to determine if perinatal IL-6 absence in these models of BPD attenuated pulmonary inflammation. IL-6 knockout (-/-) mice were compared wild-type C57BI6J mice (+/+) in order to determine if this exposure improved pulmonary inflammation. Day 14 Antepartum lipopolysaccharide (LPS) with and without postpartum hyperoxia were the two models of BPD. Litters were homogenized at birth and then randomly assigned by model type. On day 14 of life pups were sacrificed, and one third of them were randomly selected for assessment of inflammation. The primary outcome was pulmonary inflammation measured by inflammasome caspase-1 fluorometric activity at 14 days of life. Relative fluorescence units (RFU) were controlled by total protein concentration of each sample. Unpaired t-tests were utilized to determine differences between mice type for each model of BPD; a p-value < 0.05 was considered statistically significant. 23 pups were randomly selected for molecular inflammation analysis. In the LPS only model, 8 pups were analyzed. In the LPS and hyperoxia model, 15 pups were analyzed. There was no significant decrease in pulmonary inflammation between -/- and +/+ in the LPS only group (1213 vs 920 RFU/total protein, p=0.38). Additionally, there was no difference in pulmonary inflammation between the -/- and +/+ mice in the LPS and hyperoxia group (1650 vs 1559 RFU/total protein, p=0.76). Genetic IL-6 absence did not decrease pulmonary inflammation in two mouse models of BPD. Future studies should examine if a more physiologic imperfect IL-6 blockade will decrease pulmonary inflammation, due to IL-6’s roles in both the resolution of innate immunity and the development of acquired immunity.

Ultra-endurance triathlon performance and markers of whole-body and gut-specific inflammation.

To examine the influence of the Ultraman Florida triathlon (3days of non-continuous racing; stage 1: 10km swim and 144.8km cycle; stage 2: 275.4km cycle; stage 3: 84.4km run) on circulating plasma concentrations of whole-body (C-reactive protein (CRP), interleukin (IL)-6 (IL-6), and IL-10 and surrogate gut-specific inflammatory markers (IL-17 and IL-23), and determine whether these variables are associated with performance. Eighteen triathletes (N = 18; 15 men, 3 women; age: 37 ± 8 yrs) were evaluated at baseline and post-race for circulating concentrations of CRP, IL-6, IL-10, IL-17, and IL-23. Blood samples were drawn two days prior to stage 1 (1600h) and one day after stage 3 (1200h). Plasma CRP significantly increased from baseline (1985.8 ± 5962.3ng/mL) to post-race (27,013.9 ± 12,888.8ng/mL, p < 0.001, 13-fold increase). Both plasma IL-6 and IL-10 did not significantly change from baseline to post-race. Baseline and post-race concentrations of IL-17 and IL-23 were below detectable limits. Pearson's correlation between mean finish time and post-race IL-10 revealed a significant positive correlation (r = 0.54, p < 0.05). Our results suggest that cytokines such as IL-6 and IL-10 involved in the inflammatory response return to near-baseline concentrations rapidly even after ultra-endurance events of extreme duration. The absence of IL-17 and IL-23 may suggest positive gut adaptations from ultra-endurance training. A significant positive correlation between post-race IL-10 concentrations and mean finish time may indicate that a relationship between anti-inflammatory responses and performance exists.

The IL-12- and IL-23-Dependent NK Cell Response Is Essential for Protective Immunity against Secondary Toxoplasma gondii Infection.

NK cells can develop cell-intrinsic memory-like characteristics. Whether they develop these characteristics during Toxoplasma gondii infection is unknown. We addressed this question and dissected the mechanisms involved in secondary NK cell responses using a vaccine-challenge mouse model of T. gondii infection. NK cells were required for control of and survival after secondary T. gondii infection. NK cells increased in number at the reinfection site and produced IFN-γ. To test if these T. gondii experienced NK cells were intrinsically different from naive NK cells, we performed NK cell adoptive transfer into RAG2/cγ-chain-/- mice, NK cell fate mapping, and RAG1-/- mice vaccine-challenge experiments. Although NK cells contributed to immunity after reinfection, they did not develop cell-intrinsic memory-like characteristics after T. gondii vaccination. The mechanisms required for generating these secondary NK cell responses were investigated. Secondary NK cell responses were CD4+ or CD8+ T cell independent. Although IL-12 alone is required for NK cell IFN-γ production during primary T. gondii infection, in the absence of IL-12 using IL-12p35-/- mice or anti-IL-12p70, secondary NK cell responses were only partially reduced after reinfection. IL-23 depletion with anti-IL-23p19 in vivo also significantly reduced the secondary NK cell response. IL-12 and IL-23 blockade with anti-IL-12p40 treatment completely eliminated secondary NK cell responses. Importantly, blockade of IL-12, IL-23, or both significantly reduced control of parasite reinfection and increased parasite burden. Our results define a previously unknown protective role for NK cells during secondary T. gondii infection that is dependent on IL-12 and IL-23.