IL-2 Receptor Expression Research Articles

Valproic acid inhibits interferon-\u03b3 production by NK cells and increases susceptibility to Listeria monocytogenes infection

Valproic acid (VPA) is a drug commonly used for epileptic seizure control. Recently, it has been shown that VPA alters the activation of several immune cells, including Natural Killer (NK) cells, which play an important role in the containment of viruses and intracellular bacteria. Although VPA can increase susceptibility to extracellular pathogens, it is unknown whether the suppressor effect of VPA could affect the course of intracellular bacterial infection. This study aimed to evaluate the role of VPA during Listeria monocytogenes (L.m) infection, and whether NK cell activation was affected. We found that VPA significantly augmented mortality in L.m infected mice. This effect was associated with increased bacterial load in the spleen, liver, and blood. Concurrently, decreased levels of IFN-γ in serum and lower splenic indexes were observed. Moreover, in vitro analysis showed that VPA treatment decreased the frequency of IFN-γ-producing NK cells within L.m infected splenocytes. Similarly, VPA inhibited the production of IFN-γ by NK cells stimulated with IL-12 and IL-18, which is a crucial system for early IFN-γ production in listeriosis. Finally, VPA decreased the phosphorylation of STAT4, p65, and p38, without affecting the expression of IL-12 and IL-18 receptors. Altogether, our results indicate that VPA increases the susceptibility to Listeria monocytogenes infection and suggest that NK cell is one of the main targets of VPA, but further work is needed to ascertain this effect.

Serial Systemic Injections of Endotoxin (LPS) Elicit Neuroprotective Spinal Cord Microglia through IL-1-Dependent Cross Talk with Endothelial Cells

Microglia are dynamic immunosurveillance cells in the CNS. Whether microglia are protective or pathologic is context dependent; the outcome varies as a function of time relative to the stimulus, activation state of neighboring cells in the microenvironment or within progression of a particular disease. Although brain microglia can be "primed" using bacterial lipopolysaccharide (LPS)/endotoxin, it is unknown whether LPS delivered systemically can also induce neuroprotective microglia in the spinal cord. Here, we show that serial systemic injections of LPS (1 mg/kg, i.p., daily) for 4 consecutive days (LPSx4) consistently elicit a reactive spinal cord microglia response marked by dramatic morphologic changes, increased production of IL-1, and enhanced proliferation without triggering leukocyte recruitment or overt neuropathology. Following LPSx4, reactive microglia frequently contact spinal cord endothelial cells. Targeted ablation or selective expression of IL-1 and IL-1 receptor (IL-1R) in either microglia or endothelia reveal that IL-1-dependent signaling between these cells mediates microglia activation. Using a mouse model of ischemic spinal cord injury in male and female mice, we show that preoperative LPSx4 provides complete protection from ischemia-induced neuron loss and hindlimb paralysis. Neuroprotection is partly reversed by either pharmacological elimination of microglia or selective removal of IL-1R in microglia or endothelia. These data indicate that spinal cord microglia are amenable to therapeutic reprogramming via systemic manipulation and that this potential can be harnessed to protect the spinal cord from injury.SIGNIFICANCE STATEMENT Data in this report indicate that a neuroprotective spinal cord microglia response can be triggered by daily systemic injections of LPS over a period of 4 d (LPSx4). The LPSx4 regimen induces morphologic transformation and enhances proliferation of spinal cord microglia without causing neuropathology. Using advanced transgenic mouse technology, we show that IL-1-dependent microglia-endothelia cross talk is necessary for eliciting this spinal cord microglia phenotype and also for conferring optimal protection to spinal motor neurons from ischemic spinal cord injury (ISCI). Collectively, these novel data show that it is possible to consistently elicit spinal cord microglia via systemic delivery of inflammogens to achieve a therapeutically effective neuroprotective response against ISCI.

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency

Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.

MYELOID DERIVED SUPPRESSOR CELLS (MDSC) IN KIDNEY RECIPIENTS: IDENTIFICATION OF CD25+EMDSC, AND AUGMENTATION OF MONOCYTIC-MDSC EARLY AFTER TRANSPLANTATION IN PATIENTS LATER DEVELOPING CANCER

Background: MDSC increase in cancer and associate with poor prognosis, however their role in transplantation (Tx) is unknown. We aimed to study the MDSC effects on the evolution of kidney transplant recipients (KTR). Methods: A cohort of229 KTR was prospectively studied at pre- and day 7 and 14, month 1, 3, 6, and 12 post-Tx.Myeloid cells CD11b+CD33+CD14+CD15-HLA-DR-/low(M-MDSC), CD33+CD11b+CD14-HLA-DRlow/-(early stage MDSC, eMDSC) and CD11b+CD33+CD14+CD15-HLA-DR+(monocytes) were enumerated, and their suppressive capacity was tested in cocultures with autologous lymphocytes. Anti-HLA antibodies, total antioxidant capacity (TAC), arginase-1 (Arg-1), matrix metalloproteinase 9 (MMP9) and suppressive cytokines were measured in serum and supernatants. In some experiments, IL2 receptor (IL2R) expression of chains α (CD25), β (CD122) and γ (CD132) and STAT5 phosphorylation (pSTAT5) after incubation with IL2 were tested. The induction of myeloid cell death by basiliximab (Bx, anti-CD25) after adding complement (C´) was also analyzed. Results: Before Tx, HLA-DR-/low and HLA-DR+ myeloid subsets were similar among KTR and healthy volunteers. After Tx, CD11b+CD33+CD14+CD15-HLA-DR-/lowcells expanded and remained high for the first year. In vitro, these cells inhibited CD4 and CD8 T lymphocytes proliferation and IFNγ, TNFα and GM-CSF synthesis, thus confirming them as M-MDSC. The expansion of M-MDSC was not related with post-Tx events excepting for the development of cancer. Patients with M-MDSC>45.2% and >179.2 cell/µl at post-Tx day 14 had a significantly lower cancer-free survival in the 2 years after Tx (p<0.05), and showed a higher risk for cancer than patients with low M-MDSC (p=0.029 and 0.031, respectively). Multivariate Cox regression showed that M-MDSC>179.2 cell/µl was a risk factor for cancer in KTR independent of age, sex and immunosuppression (HR=6.98, p=0.02). Circulating M-MDSCs high counts correlated with high Arg-1 (p<0.01) and IL-10 (p=0.01), and low TAC (p=0.03) in serum. M-MDSC were lower in patients with enhanced alloimmune response as represented by anti-HLA sensitization. eMDSC levels also increased after Tx, excepting in patients who received Bx. CD25+eMDSC counts and the CD25per cell expression (MFI CD25) increased after Tx, but were not detected in patients who received Bx. eMDSC were found to express all three IL2R chains (high affinity IL2R), while M-MDSC and monocytes only expressed β and γ (intermediate affinity IL2R). After incubation of eMDSC with IL2, pSTAT5 was detected. Preincubation with Bx blocked both detection of CD25 and pSTAT5, but did not result in cell death after adding C´. Conclusions: Early post-transplant mobilisation of M-MDSC predicts cancer and adds risk as an independent factor in KTR. M-MDSC may favor an immunosuppressive environment that promotes the tumoral development. A CD25+eMDSC subset has been identified which is under study. ISCIII project 45/2013. CAM project B2017/BMD3731. AECC project IDEAS206PAZ.

HSV-1 Modulates IL-6 Receptor Expression on Human Dendritic Cells.

Dendritic cells (DCs) are the guardians of the immune system since they are located in the majority of peripheral tissues. In addition, they are crucial for the induction of an effective immune response based on their unique capacity to stimulate naive T cells. During co-evolution, the human pathogen herpes simplex virus type 1 (HSV-1) has evolved several immune evasion mechanisms in order to subvert the host's immune system especially by targeting DC biology and function. Here we demonstrate that HSV-1 infection influences the IL-6 receptor (IL6R) expression both on protein and mRNA levels in/on human monocyte-derived mature DCs (mDCs). Surprisingly, reduced IL6R expression levels were also observed on uninfected bystander mDCs. Mechanistically, we clearly show that HSV-1-derived non-infectious light (L-) particles are sufficient to trigger IL6R regulation on uninfected bystander mDCs. These L-particles lack the viral DNA-loaded capsid and are predominantly produced during infection of mDCs. Our results show that the deletion of the HSV-1 tegument protein vhs partially rescued the reduced IL6R surface expression levels on/in bystander mDCs. Using a neutralizing antibody, which perturbs the transfer of L-particles to bystander mDCs, was sufficient to rescue the modulation of IL6R surface expression on uninfected bystander mDCs. This study provides evidence that L-particles transfer specific viral proteins to uninfected bystander mDCs, thereby negatively interfering with their IL6R expression levels, however, to a lesser extend compared to H-particles. Due to their immune-modulatory capacity, L-particles represent an elaborated approach of HSV-1-mediated immune evasion.

Delivering IL-2 to Tregs

Cytokines Interleukin-2 (IL-2) not only promotes activation and expansion of conventional T cells but also plays a crucial role in regulating homeostasis and functions of regulatory T cells (Tregs). Because expression of the high-affinity IL-2 receptor is largely restricted to Tregs, engineering IL-2 variants called muteins that selectively bind to the high-affinity IL-2 receptor and potentiate Treg functions can be used in the treatment of autoimmune diseases. Khoryati et al. engineered a mouse IL-2 mutein that selectively promotes expansion of Tregs. They demonstrate that administration of this IL-2 mutein resolved ongoing diabetes in nonobese diabetic mice. This proof-of-principle study raises the possibility that engineered human IL-2 muteins could be used in the treatment of autoimmune diseases. Sci. Immunol. 5 , eaba5264 (2020).

The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer.

The cytokine IL22 promotes tumor progression in murine models of colorectal cancer. However, the clinical significance of IL22 in human colorectal cancer remains unclear. We sought to determine whether the IL22 pathway is associated with prognosis in human colorectal cancer, and to identify mechanisms by which IL22 can influence disease progression. Transcriptomic data from stage II/III colon cancers in independent discovery (GSE39582 population-based cohort, N = 566) and verification (PETACC3 clinical trial, N = 752) datasets were used to investigate the association between IL22 receptor expression (encoded by the genes IL22RA1 and IL10RB), tumor mutation status, and clinical outcome using Cox proportional hazard models. Functional interactions between IL22 and mutant KRAS were elucidated using human colorectal cancer cell lines and primary tumor organoids. Transcriptomic analysis revealed a poor-prognosis subset of tumors characterized by high expression of IL22RA1, the alpha subunit of the heterodimeric IL22 receptor, and KRAS mutation [relapse-free survival (RFS): HR = 2.93, P = 0.0006; overall survival (OS): HR = 2.45, P = 0.0023]. KRAS mutations showed a similar interaction with IL10RB and conferred the worst prognosis in tumors with high expression of both IL22RA1 and IL10RB (RFS: HR = 3.81, P = 0.0036; OS: HR = 3.90, P = 0.0050). Analysis of human colorectal cancer cell lines and primary tumor organoids, including an isogenic cell line pair that differed only in KRAS mutation status, showed that IL22 and mutant KRAS cooperatively enhance cancer cell proliferation, in part through augmentation of the Myc pathway. Interactions between KRAS and IL22 signaling may underlie a previously unrecognized subset of clinically aggressive colorectal cancer that could benefit from therapeutic modulation of the IL22 pathway.

Abstract 1597: The landscape of PD1-PD-L1 axis in the context of immune cells: Tumors versus stromal compartments

Abstract Introduction: Immune response, tolerance, suppression, and resistance are the result of a complex dynamic interaction comprising PD1-PD-L1 axis in the context of immune cells (ICs) involving tumor cells, and anti/pro-tumor immune-cells of the stromal compartment. Aim: Here we present the landscape of PD1-PD-L1 axis in the context of CD3, CD4, CD8, PD1, FOXP3, and IL12RB2 positive ICs in the tumor (T) and the tumor-adjacent normal (TAN) tissues from patients with ovarian cancers (OC), and lung cancers (LC). Methods: The study was approved by Avera IRB. Surgically resected paired T and TAN tissue samples (from patients with OC and LC) were used to identify CD3 (ab16669), CD4 (ab133616), CD8 (ab85792), FOXP3 ([236A/E7], PD1 (ab137132), IL12RB2 (NBP1-85983), CD68 (Dako. #M0876), CD163 (Cell Signaling #93498) in ICs and PD-L1 (Clone 22C3; Dako) in T cells &amp; tumor-associated macrophages (TAMs). Tonsil tissue was used for the IHC validation. Paired specimens were morphologically evaluated by H&amp;E stain &amp; Ki-67, cl-caspase 3. Anatomic pathological evaluation for the diagnosis of the disease was carried out by a Pathologist. Results: Expression of CD3, CD4, CD8, PD1, and FOXP3 was heterogeneous, with each tumor presenting an individual pattern of distribution of ICs. However in most tumor samples, the distribution of ICs was of tumor-cooperating phenotypes. PD-L1 expression in tumors did not correlate with any other ICs of the tumor-microenvironment. In TAN tissues from OC, PD1-PD-L1 staining was absent. While macrophages in TAN tissue from LC were moderately positive for PD-L1. In T tissue from OC and LC, PD1-PD-L1 staining was only present in the stroma, lymphocytes, and absent in the tumor compartments while PD-L1 staining was identified only in the macrophages. Tumoricidal cells were rarely observed within the tumor compartment of T tissues from OC. The tumors cells from LC was only positive for PD-L1. Conclusion: Using dual stain of PD1 and PD-L1, we present for the first time, the co-distribution pattern of PD1-PD-L1 axis in the context of the expression of CD3, CD4, CD8, FOXP3 T cells as well as CD68, and CD163 positive TAMs in the paired sample of a T tissue and its TAN tissue in LC and OC. The relationship between the PD1-PD-L1 axis and the expression of IL12 Receptor in the context of immune-landscape is being worked out and will be presented in the meeting. Citation Format: Xiaoqian Lin, Raed Sulaiman, Jennifer C. Aske, Paul Meyer, Luis RojasEspaillat, David Starks, Pradip De, Nandini Dey. The landscape of PD1-PD-L1 axis in the context of immune cells: Tumors versus stromal compartments [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 1597.

Abstract 3368: A novel anti-CTLA-4 antibody with potent anti-tumor activity emerged using humanized CTLA-4 mouse model

Abstract CTLA-4 (Cytotoxic T-lymphocyte antigen 4), also known as CD152, belongs to immunoglobulin superfamily. It is an intracellular protein expressed in resting T cells; CTLA-4 translocates to the cell surface and transmits an inhibitory signal to T cells. In addition, CTLA-4 competes with its homologous T-cell co-stimulatory protein CD28 for ligands expressed on APC, namely CD80 (B7-1) and CD86 (B7-2), thereby antagonizing CD28 and inhibiting CD28 signaling, resulting in inhibition of IL-2, IFN-γ, IL-4 cytokines production, IL-2 receptor expression and cell cycle progression. Because Tregs constitutively express CTLA-4 at high levels, anti-CTLA-4 therapy would be expected to have the greatest effect on these cells. Ipilimumab, a fully human IgG1 monoclonal antibody, is the only commercially available CTLA-4 antagonist. It has been approved by FDA for treatment of metastatic melanoma, adjuvant melanoma and advanced renal cell carcinoma, based on its significant effect of tumor suppression demonstrated in clinical trials. We have interests in searching for potent CTLA-4 antagonists. We selected an antibody with the best efficacy, namely YH001, from the candidate molecules through Biocytoge's in vivo drug screening platform. YH001 is a recombinant humanized anti-CTLA-4 IgG1 monoclonal antibody and it can bind with human CTLA-4 potently and specifically, without reactivity with other CD28 family receptors. YH001 was able to block the CTLA-4/CD80 and CTLA-4/CD86 interactions and potentiate CD28 signaling in the presence of cognate TCR stimulation. Moreover, YH001 also induced stronger bioluminescence than ipilimumab in the reporter assay. YH001 showed more potent ADCC activities than ipilimumab. YH001 showed dose-dependent in vivo anti-cancer efficacy, as well as Treg depletion and increased CD8+/Treg ratio in human CTLA-4 knock-in mice bearing MC38 tumors. Exposure to YH001 was dose proportional with a half-life of approximately 5 days in cynomolgus monkey. Our preclinical investigation of YH001 demonstrates Biocytogen's capability in discovering clinical candidates using its robust antibody discovery platform. Citation Format: Chaoshe Guo, Yi Yang, Zhihong Li, Yanan Li, Yunyun Chen. A novel anti-CTLA-4 antibody with potent anti-tumor activity emerged using humanized CTLA-4 mouse model [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 3368.

Immune-enhancing effects of glucocorticoids in response to day-night cycles and stress.

Environmental cues such as the day-night cycle or stressors trigger the production of glucocorticoids (GCs) by the adrenal cortex. GCs are well known for their anti-inflammatory effects that suppress the production of inflammatory cytokines and induce the apoptosis of lymphocytes. Recent studies in mice, however, have revealed pro-inflammatory effects. The diurnal oscillation of GCs induces the expression of IL-7 receptor α (IL-7Rα) and C-X-C motif chemokine receptor 4 (CXCR4) at the active phase, which drives the diurnal homing of T cells into lymphoid organs. This accumulation of T cells at the active phase enhances T-cell priming against bacterial infection and antigen immunization, leading to an increase of effector CD8 T cells and antibody production. GCs induced by moderate stress trigger the homing of memory CD8 T cells into the bone marrow and support the maintenance and response of these cells. Thus, endogenous GCs have a self-defense function to enhance adaptive immune responses. By contrast, strong stress induces even higher GC levels and causes chronic inflammation and autoimmunity. Because GCs can enhance the differentiation and function of T-helper 2 (Th2) and Th17 cells, high stress-induced GC levels might enhance inflammation via Th17 cell differentiation. Overall, the positive and negative effects of GCs may regulate the balance between normal immune responses and susceptibility to infections and inflammatory diseases.

Innate Lymphocyte Th1 and Th17 Responses in Elderly Hospitalised Patients with Infection and Sepsis.

Background: the role of innate immunity in human sepsis must be fully clarified to identify potential avenues for novel immune adjuvant sepsis therapies. Methods: A prospective observational study was performed including patients with sepsis (septic group), infection without sepsis (infection group), and healthy controls (control group) in the setting of acute medical wards and intensive care units in a 1000-bed university hospital. A total of 42 patients with sepsis, 30 patients with infection, and 30 healthy controls were studied. The differentiation states of circulating mucosal associated invariant T (MAIT) cells and Natural Killer T (NKT) cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA−, CD197+), effector memory (CD45RA−, CD197−), or terminally differentiated (CD45RA+, CD197−). The differentiation states of circulating gamma-delta T lymphocytes were characterised as naive (CD45RA+, CD27+), central memory (CD45RA−, CD27+), effector memory (CD45RA−, CD27−), or terminally differentiated (CD45RA+, CD27−). The expression of IL-12 and IL-23 receptors, the transcription factors T-Bet and RORγt, and interferon-γ and IL-17a were analysed. Results: MAIT cell counts were lower in the septic group (p = 0.002) and the infection group (p < 0.001) than in the control group. The MAIT cell T-Bet expression in the infection group was greater than in the septic group (p = 0.012). The MAIT RORγt expression in the septic group was lower than in the control group (p = 0.003). The NK cell counts differed in the three groups (p < 0.001), with lower Natural Killer (NK) cell counts in the septic group (p < 0.001) and in the infection group (p = 0.001) than in the control group. The NK cell counts increased in the septic group in the 3 weeks following the onset of sepsis (p = 0.028). In lymphocyte stimulation experiments, fewer NK cells expressed T-Bet in the septic group than in the infection group (p = 0.002), and fewer NK cells expressed IFN-γ in the septic group than in the control group (p = 0.002). The NKT cell counts were lower in the septic group than both the control group (p = 0.05) and the infection group (p = 0.04). Fewer NKT cells expressed T-Bet in the septic group than in the infection group (p = 0.004). Fewer NKT cells expressed RORγt in the septic group than in the control group (p = 0.003). Fewer NKT cells expressed IFN-γ in the septic group than in both the control group (p = 0.002) and the infection group (p = 0.036). Conclusion: The clinical presentation of infection and or sepsis in patients is linked with a mosaic of changes in the innate lymphocyte Th1 and Th17 phenotypes. The manipulation of the innate lymphocyte phenotype offers a potential avenue for immune modulation in patients with sepsis.

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self

Background aimsNatural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells. MethodsPC3PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells. ResultsOnly combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen. ConclusionsCollectively, the results demonstrate the feasibility of PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.

Hypercapnia exacerbates the disruption of the blood‑brain barrier by inducing interleukin‑1β overproduction in the blood of hypoxemic adult rats.

Refractory hypoxemia is the main symptom of acute respiratory distress syndrome (ARDS). Low tidal volume ventilation is routinely applied in clinical practice to correct hypoxemia, which aims to prevent ventilator-induced lung injury. However, this ventilation strategy inevitably leads to hypercapnia. Our previous study demonstrated that hypercapnia aggravated cognitive impairment in hypoxemic rats; however, the underlying mechanism remains unclear. The aim of the present study was to investigate whether hypercapnia exacerbates the blood-brain barrier (BBB) disruption through inducing interleukin (IL)-1β overproduction in the blood of hypoxemic rats. The BBB permeability in a rat model of hypercapnia/hypoxemia was evaluated. The levels of IL-1β in the blood of rats and human whole-blood cultures were assessed. The expression of IL-1 receptor 1 (IL-1R1), phosphorylated IL-1R1-associated kinase (p-IRAK-1) and tight junctional proteins in cerebral vascular endothelial cells was examined in vitro and in vivo. In addition, IL-1Ra, an IL-1 receptor antagonist, was used to determine whether hypercapnia affects tight junctional protein expression in hypoxic cerebral vascular endothelial cells through inducing IL-1β overproduction. It was observed that hypercapnia alone did not disrupt the BBB, but aggravated the damage to the BBB integrity in hypoxemic rats. Hypercapnia increased IL-1β expression in the blood of hypoxemic rats as well as in hypoxic human whole-blood cultures. IL-1R1 and p-IRAK-1 expression was increased, while that of tight junctional proteins was reduced by hypercapnia in hypoxemic cerebral vascular endothelial cells in vitro and in vivo. Additionally, the expression of tight junctional proteins was markedly increased following treatment with IL-1Ra. These results suggest that hypercapnia-induced IL-1β overproduction in the hypoxemic blood may decrease tight junctional protein expression in cerebrovascular endothelial cells via the IL-1R1/p-IRAK-1 pathway, further disrupting BBB integrity, and eventually resulting in increased BBB permeability.

Characterization of the interleukin-17 effect on articular cartilage in a translational model: an explorative study

BackgroundOsteoarthritis (OA) is a progressive, chronic disease characterized by articular cartilage destruction. The pro-inflammatory cytokine IL-17 levels have been reported elevated in serum and synovial fluid of OA patients and correlated with increased cartilage defects and bone remodeling. The aim of this study was to characterize an IL-17-mediated articular cartilage degradation ex-vivo model and to investigate IL-17 effect on cartilage extracellular matrix protein turnover.MethodsFull-depth bovine femoral condyle articular cartilage explants were cultured in serum-free medium for three weeks in the absence, or presence of cytokines: IL-17A (100 ng/ml or 25 ng/ml), or 10 ng OSM combined with 20 ng/ml TNFα (O + T). RNA isolation and PCR analysis were performed on tissue lysates to confirm IL-17 receptor expression. GAG and ECM-turnover biomarker release into conditioned media was assessed with dimethyl methylene blue and ELISA assays, respectively. Gelatin zymography was used for matrix metalloproteinase (MMP) 2 and MMP9 activity assessment in conditioned media, and shotgun LC-MS/MS for identification and label-free quantification of proteins and protein fragments in conditioned media. Western blotting was used to validate MS results.ResultsIL-17RA mRNA was expressed in bovine full-depth articular cartilage and the treatment with IL-17A did not interfere with metabolic activity of the model. IL-17A induced cartilage breakdown; conditioned media GAG levels were 3.6-fold-elevated compared to untreated. IL-17A [100 ng/ml] induced ADAMTS-mediated aggrecan degradation fragment release (14-fold increase compared to untreated) and MMP-mediated type II collagen fragment release (6-fold-change compared to untreated). MS data analysis revealed 16 differentially expressed proteins in IL-17A conditioned media compared to untreated, and CHI3L1 upregulation in conditioned media in response to IL-17 was confirmed by Western blotting.ConclusionsWe showed that IL-17A has cartilage modulating potential. It induces collagen and aggrecan degradation indicating an upregulation of MMPs. This was confirmed by zymography and mass spectrometry data. We also showed that the expression of other cytokines is induced by IL-17A, which provide further insight to the pathways that are active in response to IL-17A. This exploratory study confirms that IL-17A may play a role in cartilage pathology and that the applied model may be a good tool to further investigate it.

Interleukin-9 regulates macrophage activation in the progressive multiple sclerosis brain

BackgroundMultiple sclerosis (MS) is an immune-mediated, chronic inflammatory, and demyelinating disease of the central nervous system (CNS). Several cytokines are thought to be involved in the regulation of MS pathogenesis. We recently identified interleukin (IL)-9 as a cytokine reducing inflammation and protecting from neurodegeneration in relapsing–remitting MS patients. However, the expression of IL-9 in CNS, and the mechanisms underlying the effect of IL-9 on CNS infiltrating immune cells have never been investigated.MethodsTo address this question, we first analyzed the expression levels of IL-9 in post-mortem cerebrospinal fluid of MS patients and the in situ expression of IL-9 in post-mortem MS brain samples by immunohistochemistry. A complementary investigation focused on identifying which immune cells express IL-9 receptor (IL-9R) by flow cytometry, western blot, and immunohistochemistry. Finally, we explored the effect of IL-9 on IL-9-responsive cells, analyzing the induced signaling pathways and functional properties.ResultsWe found that macrophages, microglia, and CD4 T lymphocytes were the cells expressing the highest levels of IL-9 in the MS brain. Of the immune cells circulating in the blood, monocytes/macrophages were the most responsive to IL-9. We validated the expression of IL-9R by macrophages/microglia in post-mortem brain sections of MS patients. IL-9 induced activation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 and reduced the expression of activation markers, such as CD45, CD14, CD68, and CD11b in inflammatory macrophages stimulated in vitro with lipopolysaccharide and interferon (IFN)-γ. Similarly, in situ the number of activated CD68+ macrophages was significantly reduced in areas with high levels of IL-9. Moreover, in the same conditions, IL-9 increased the secretion of the anti-inflammatory cytokine, transforming growth factor (TGF)-β.ConclusionsThese results reveal a new cytokine expressed in the CNS, with a role in the context of MS. We have demonstrated that IL-9 and its receptor are both expressed in CNS. Moreover, we found that IL-9 decreases the activation state and promotes the anti-inflammatory properties of human macrophages. This mechanism may contribute to the beneficial effects of IL-9 that are observed in MS, and may be therapeutically potentiated by modulating IL-9 expression in MS.

Age-related changes in the immune phenotype of human memory CD4 T cells differ between colon and peripheral blood

Abstract Gut lamina propria (LP) CD4 T cells are primarily tissue resident memory cells that are critical in maintaining gut homeostasis and responding to enteric pathogens. We established that aging altered the phenotypic and functional profiles of human small intestinal CD4 T cells. However, given site-specific differences in immune cell composition in the gut, it is difficult to extrapolate these findings to other locations. To probe the effect of aging on large intestine CD4 T cells, we used flow cytometry to analyze immune phenotypes of colon LP CD4 T cells from younger (34±2yrs mean±SEM; N=6) and older (80±2yrs, N=6) adults. Tissue-specificity of these profiles was determined by comparing to memory (CD45RA−) peripheral blood (mPB) CD4 T cells in a separate cohort of similarly aged persons (N=6/age group). Among younger samples, LP CD4 T cells expressed higher levels of co-inhibitory molecules CTLA4 and PD1, but had lower turnover (Ki67+) and IL2 receptor (CD25) expression relative to mPB CD4 T cells. Expression of activation (CD38, HLA-DR), senescence (CD57) and survival (Bcl2) markers were similar. Regarding age effects, frequencies of LP CD4 T cells (% of CD3 T cells) were similar between older and younger samples; mPB CD4 T cell frequencies were higher in older vs younger samples. Older LP CD4 T cells had lower CTLA4, PD1, CD38, CD25 and Ki67 and higher Bcl2 expression than younger LP CD4 T cells. Among mPB CD4 T cells, greater age was only associated with a higher CD57+ fraction. Thus, age effects differed between colon LP and mPB CD4 memory T cells. Loss of co-inhibitory markers, decreased turnover and increased survival of colon LP CD4 T cells may contribute to altered homeostasis, loss of protective immunity, and chronic inflammation in older people.

Intestinal colonization with IL-10 inducing adherent E. coli protects from intestinal inflammation and tumor development

Abstract Colorectal cancer (CRC) is the third most common and third most deadly cancer in the US. Chronic intestinal inflammation corresponds with advanced disease stage and decreased survival in CRC. Intestinal inflammation corresponds with changes in the microbiota, including expanded proteobacteria, such as E. coli. This altered microbiota is thought to support inflammation and disease progression in both CRC and inflammatory bowel disease (IBD). However, how individual microbes interact with host cells and the pathways they activate remains incompletely defined. We characterized immune effects after colonization with individual isolates from a panel of human mucosally associated E. coli. In contrast to our expectations, a subset induced intestinal macrophage production of IL-10, protecting from colitis. Further characterization of a single isolate revealed colonization with this isolate limited tumor incidence in a model of inflammation associated colorectal tumors. Intestinal macrophages, macrophage production of IL-10, and epithelial expression of IL-10 receptor were all required for anti-inflammatory and anti-tumor effects. Colonization with an isogenic mutant E. coli that lacked a single gene important for epithelial attachment did not induce IL-10 and amplified intestinal tumors with increased tumor infiltration of anti-inflammatory immune cells. While the inherent diversity of the microbiota makes it difficult to pinpoint singular pathways of tumorigenesis, we have identified a regulatory loop between adherent microbes, intestinal epithelium and intestinal immune cells which promotes barrier integrity and limits tumor development, identifying novel ways to therapeutically target inflammation and CRC.

The Th9 Axis Reduces the Oxidative Stress and Promotes the Survival of Malignant T Cells in Cutaneous T-Cell Lymphoma Patients.

Immune dysfunction is critical in pathogenesis of cutaneous T-cell lymphoma (CTCL). Few studies have reported abnormal cytokine profile and dysregulated T-cell functions during the onset and progression of certain types of lymphoma. However, the presence of IL9-producing Th9 cells and their role in tumor cell metabolism and survival remain unexplored. With this clinical study, we performed multidimensional blood endotyping of CTCL patients before and after standard photo/chemotherapy and revealed distinct immune hallmarks of the disease. Importantly, there was a higher frequency of "skin homing" Th9 cells in CTCL patients with early (T1 and T2) and advanced-stage disease (T3 and T4). However, advanced-stage CTCL patients had severely impaired frequency of skin-homing Th1 and Th17 cells, indicating attenuated immunity. Treatment of CTCL patients with standard photo/chemotherapy decreased the skin-homing Th9 cells and increased the Th1 and Th17 cells. Interestingly, T cells of CTCL patients express IL9 receptor (IL9R), and there was negligible IL9R expression on T cells of healthy donors. Mechanistically, IL9/IL9R interaction on CD3+ T cells of CTCL patients and Jurkat cells reduced oxidative stress, lactic acidosis, and apoptosis and ultimately increased their survival. In conclusion, coexpression of IL9 and IL9R on T cells in CTCL patients indicates the autocrine-positive feedback loop of Th9 axis in promoting the survival of malignant T cells by reducing the oxidative stress. IMPLICATIONS: The critical role of Th9 axis in CTCL pathogenesis indicates that strategies targeting Th9 cells might harbor significant potential in developing robust CTCL therapy.

Lysosomotropic drugs enhance pro-inflammatory responses to IL-1β in macrophages by inhibiting internalization of the IL-1 receptor

Interleukin (IL)-1 signaling leads to production of pro-inflammatory mediators and is regulated by receptor endocytosis. Lysosomotropic drugs have been linked to increased pro-inflammatory responses under sterile inflammatory conditions but the underlying mechanisms have not been fully elucidated. Here, we report that lysosomotropic drugs potentiate pro-inflammatory effects in response to IL-1β via a mechanism involving reactive oxygen species, p38 mitogen-activated protein kinase and reduced IL-1 receptor internalization. Chloroquine and hydroxychloroquine increased IL-1β-induced CXCL8 secretion in macrophages which was critically dependent on the lysosomotropic character and inhibition of macroautophagy but independent from the NLRP3 inflammasome. Co-stimulation with the autophagy inducer interferon gamma attenuated CXCL8 release. Other lysosomotropic drugs like bafilomycin A1, fluoxetine and chlorpromazine but also the endocytosis inhibitor dynasore showed similar pro-inflammatory responses. Increased cell surface expression of IL-1 receptor suggests reduced receptor degradation in the presence of lysosomotropic drugs. Our findings provide new insights into a potentially crucial immunoregulatory mechanism in macrophages that may explain how lysosomotropic drugs drive sterile inflammation.

IL-3 Receptor Expression on Activated Human Th Cells Is Regulated by IL-4, and IL-3 Synergizes with IL-4 to Enhance Th2 Cell Differentiation.

IL-3, a cytokine secreted by activated T lymphocytes, is known to regulate the proliferation, survival, and differentiation of hematopoietic cells. However, the role of IL-3 in regulation of T cell functions is not fully delineated. Previously, we have reported that IL-3 plays an important role in development of regulatory T cells in mice. In this study, we investigated the regulation of IL-3R expression on human Th cells and also examined the role of IL-3 in effector functions of these cells. We found that human peripheral blood Th cells in resting state do not show surface expression of IL-3R; however, its expression was observed at transcript and intracellular protein levels. The functional IL-3R expression on the surface was seen only after antigenic stimulation. When naive Th cells were activated in the presence of various cytokines, we found that IL-4 significantly increases the surface expression of IL-3R and also increases the number of IL-3R+ Th cells. Interestingly, IL-3R+ cells exhibit a Th2 cell-like phenotype and show high GATA-3 expression. Moreover, Th2 cells in presence of IL-3 show increased expression of type 2 effector cytokines, such as IL-4, IL-5, and IL-13. Furthermore, IL-3R expressing and IL-3-secreting Th cells were high in house dust mite-allergic patients. Thus, to our knowledge, we provide the first evidence that the expression of IL-3R on activated human Th cells is modulated by IL-4, and IL-3 regulates the effector functions of Th2 cells. Our results suggest that IL-3 may play an important role in regulating allergic immune responses.