Lymphocyte Homeostasis Research Articles

The ITK Inhibitor Cpi-818 Blocks Activation of T Cells from Autoimmune Lymphoproliferative Syndrome (ALPS) Patients and Is Active in a Murine Model of ALPS

Introduction We present preclinical evidence confirming the expression of ITK in double negative (DN) T cells of ALPS patients and demonstrate that CPI-818 inhibits the TCR-induced activation of these cells ex vivo. Furthermore, CPI-818 effectively suppressed the accumulation of DN T cells in the MRL/lpr mice, leading to dramatic improvements in lymphadenopathy, splenomegaly and other pathologies associated with lymphocyte accumulation due to Fas-deficiency. ALPS is a disorder of lymphocyte apoptosis resulting in childhood onset lymphadenopathy, multilineage cytopenias, hypersplenism and autoimmune peripheral destruction. Most ALPS cases are associated with a loss of function mutation in the Fas gene resulting in immune dysregulation such as a marked elevation of abnormal TCR+CD4-CD8-double negative (DN) T cells. ALPS patients are currently treated with immunosuppressive and cytotoxic agents, hence targeted, less toxic treatments are imperative. The critical role of Fas protein in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was initially discovered with studies in Fas-deficient MRL/LpJ-Tnfrsf6lpr (MRL/lpr) mice. These mice have dramatically elevated levels of DN T cells and develop many clinical manifestations similar to those observed in ALPS patients. Thus, MRL/lpr mice have been a useful model to assess compounds and therapeutic strategies for the treatment of ALPS. Interleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase critical to T cell receptor (TCR) signal transduction, which modulates T cell proliferation and differentiation. We sought to test whether the effect of selective inhibition of TCR signaling through ITK inhibition could be a strategy to suppress the DN T cell expansion and disease manifestation in MRL/lpr mice. CPI-818 is an orally bioavailable, covalent inhibitor of ITK that potently inhibits TCR signal transduction. It is currently being evaluated in a phase 1/1b clinical trial in T-cell lymphoma (NCT03952078) where it has been well tolerated and resulted in anti-tumor activity. Methods PBMC samples were obtained from healthy donors or ALPS patients collected under NIAID IRB-approved protocol 93-I-0063. Cells were cultured with or without anti-CD3/CD28 activation. The impact of CPI-818 on viability, expression of ITK, and expression of the surface markers CD 25 and CD69 was assessed by flow cytometry. For in vivo studies, MRL/lpr mice received a control diet or a CPI-818 formulated diet (300 mg/kg/day). Lymph nodes were calipered weekly over the course of 22 weeks of disease development. Spleens, lungs and kidneys were harvested at 22 weeks to assess the impact of CPI-818 treatment on T cell subsets and histology. Results Ex vivo studies with PBMC preparations from normal donors and ALPS patients confirmed the expression of ITK in CD4+, CD8+, and DN T cells from ALPS patients. When these T cells were stimulated with anti-CD3/CD28, CPI-818 potently inhibited the upregulation of the activation markers CD25 and CD69 in DN T cells assessed by flow cytometry. In companion experiments, CPI-818 was not cytotoxic to DN T cells when activated through the TCR signaling pathway. Treatment of MRL/lpr mice with CPI-818 prevented the onset or, in a therapeutic design, led to the regression of lymphadenopathy and splenomegaly comparable to the treatment effects observed with cyclophosphamide. Unlike the effects seen with the cytotoxic agent cyclophosphamide which suppressed all T cells, as well as B, NK and myeloid cells in the spleen, CPI-818 selectively depleted DN T cells (see Figure). CPI-818 treatment also resulted in decreased levels of DN T cells in the kidneys and lungs of these mice without impacting the level of normal CD4 and CD8 T cells. CPI-818 limited the increased proteinuria measured in the control animals during the 22-week treatment window. Histological assessment of the kidney revealed reduced inflammatory damage in CPI-818 treated animals. Conclusions The selective, covalent ITK inhibitor, CPI-818, significantly reduced DN T cell numbers, lymphadenopathy and other disease end points in the MRL/lpr murine model of ALPS. This suggests that the dysregulated growth of Fas-deficient DN T cells requires functional TCR signaling. Further, our data suggests that blocking TCR signaling by targeting ITK may be an effective strategy for treating adult and pediatric ALPS. Figure Disclosures Ng: Corvus Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Hill:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Miller:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Buggy:Corvus Pharmaceuticals: Consultancy, Current Employment, Current equity holder in publicly-traded company. Janc:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

PD-1 Expression on CD8+CD28- T cells within inflammatory synovium is associated with Relapse: A cohort of Rheumatoid Arthritis

Defect in T lymphocyte homeostasis could implicate initiation and development of rheumatoid arthritis (RA). Since PD-1 plays a key role in the regulation of T lymphocytes, its expression pattern in various CD8+ T cell subsets could be so effective in RA pathogenesis. Here, we investigated the expression of PD-1 and CXCR3 on CD8+CD28- T cells in association with the IFN-γ levels in patients with RA. A total of 42 RA patients, including 10 newly-diagnosed (ND) and 32 relapsed (RL) cases and also 20 healthy donors were enrolled. Phenotypic characterization of CD8+ T cells derived from peripheral blood (PB) and synovial fluid (SF) was performed by flow cytometry. The plasma and SF IFN-γ levels were also assessed by ELISA. The frequency of CD8+CD28- T cells showed no significant differences between patients and controls while its higher levels were observed in PB, versus SF of RL patients. Relapsed patients also showed higher CXCR3 and especially PD-1 expression on their CD8+CD28- T cells. The IFN-γ concentration was elevated in SF of ND patients while its plasma level was significantly lower in RL subgroup than controls. Although PD-1 could induce immune suppression in effector T cells, it is upregulated during inflammation and its overexpression on CD8+CD28- T cells within inflammatory synovium is associated with severity of disease in our cohort of RA patients.

The relationship between Chlamydia pneumoniae infection and CD4/CD8 ratio, lymphocyte subsets in middle-aged and elderly individuals

Chlamydia pneumoniae (C. pneumoniae) is a common respiratory pathogen associated with many inflammatory diseases. There are few data concerning the lymphocyte subsets in middle-aged and elderly individuals with C. pneumoniae infection. A total of 191 patients were included in this study. The study population was categorized into the middle-aged group (40–64 years old) and the elderly group (65–89 years old). Lymphocyte subsets in peripheral blood were examined with multi-colored flow cytometry. Immunological monitoring included lymphocyte subsets, C. pneumoniae IgG and IgM serology. In the middle-aged group, 69.83% individuals presented IgG positivity, which was associated with the inverted CD4/CD8 ratio. Individuals with C. pneumoniae IgG positivity also presented an increased percentage of CD8+CD28− cells and a decreased CD4/CD8 ratio when compared to weakly-positive individuals. In the elderly group, C. pneumoniae IgG positivity was associated with a significant increase in the percentage of CD3+CD56+CD45+ (NKT) cells. In conclusion, altered lymphocyte homeostasis was shown in middle-aged individuals with C. pneumoniae IgG positivity. The senescent phenotypes of T cells might be associated with C. pneumoniae infection in middle-aged individuals.

Control of T lymphocyte fate decisions by PI3K signaling.

Virtually all aspects of T and B lymphocyte development, homeostasis, activation, and effector function are impacted by the interaction of their clonally distributed antigen receptors with antigens encountered in their respective environments. Antigen receptors mediate their effects by modulating intracellular signaling pathways that ultimately impinge on the cytoskeleton, bioenergetic pathways, transcription, and translation. Although these signaling pathways are rather well described at this point, especially those steps that are most receptor-proximal, how such pathways contribute to more quantitative aspects of lymphocyte function is still being elucidated. One of the signaling pathways that appears to be involved in this “tuning” process is controlled by the lipid kinase PI3K. Here we review recent key findings regarding both the triggering/enhancement of PI3K signals (via BCAP and ICOS) as well as their regulation (via PIK3IP1 and PHLPP) and how these signals integrate and determine cellular processes. Lymphocytes display tremendous functional plasticity, adjusting their metabolism and gene expression programs to specific conditions depending on their tissue of residence and the nature of the infectious threat to which they are responding. We give an overview of recent findings that have contributed to this model, with a focus on T cells, including what has been learned from patients with gain-of-function mutations in PI3K as well as lessons from cancer immunotherapy approaches.

Clostridium perfringens phospholipase C impairs innate immune response by inducing integrated stress response and mitochondrial-induced epigenetic modifications

Clostridium perfringens, a rod-shaped, gram-positive, anaerobic, spore-forming bacterium is one of the most widely occurring bacterial pathogens, associated with a spectrum of diseases in humans. A major virulence factor during its infection is the enzyme phospholipase C encoded by the plc gene, known as Clostridium perfringens phospholipase C (CpPLC). The present study was designed to understand the role of CpPLC in inducing survival mechanisms and mitochondrial-induced epigenetic changes in a human lymphocyte cell culture model. Following exposure to CpPLC, a significant generation of mitochondrial reactive oxygen species was observed, which coincided with the changes in the expression of vital components of MAP/ERK/RTK signaling cascade that regulates the downstream cellular functions. These disturbances further led to alterations in the mitochondrial genome and functioning. This was supported by the observed upregulation in the expression of mitochondrial fission genes Drp1, Fis1, and Mff, and mitochondrial fusion genes MFN1, MFN2, and OPA1 following CpPLC exposure. CpPLC exposed cells showed upregulation of OMA1, DELE1, and HRI genes involved in the integrated stress response (ISR), which suggests that it may induce the ISR that provides a pro-survival mechanism to the host cell. CpPLC also initiated immune patho-physiologic mechanisms including mitochondrial-induced epigenetic modifications through a mitochondrial-ROS driven signaling pathway. Interestingly, epigenetic machinery not only play a pivotal role in lymphocyte homeostasis by contributing to cell-fate decisions but thought to be one of the mechanisms by which intracellular pathogens survive within the host cells. Importantly, the impairment of mtDNA repair among the CpPLC exposed cells, induced alterations within mtDNA methylation, and led to the deregulation of MT-CO1, MT-ND6, MT-ATPase 6, and MT-ATPase8 gene expression profiles that are important for mitochondrial bioenergetics and subsequent metabolic pathways. This was further confirmed by the changes in the activity of mitochondrial electron chain complexes (complex I, II, III, IV and V). The altered mtDNA methylation profile was also found to be closely associated with the varied expression of mitomiRs and their targets. CpPLC exposed cells showed up-regulation of miR24 expression and down-regulation of miR34a, miR150, and miR155, while the increased expression of mitomiR target genes i.e. of K-Ras, MYC, EGFR, and NF-kβ was also observed in these cells. Altogether, our findings provide novel insights into the derailment of redox signaling machinery in CpPLC treated lymphocytes and its role in the induction of survival mechanisms and mitochondrial-induced epigenetic modifications.

The clinical value of the changes of peripheral lymphocyte subsets absolute counts in patients with non-small cell lung cancer.

IntroductionImmune function strongly influences the outcome of patients with non-small cell lung cancer (NSCLC). It's vital to understand the immune state of patients through detecting the percentage and number of lymphocyte subsets accurately, and helpful to evaluate conditions of prognosis and adjust treatment for patients.MethodsWe conducted a retrospective cohort study in First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China. The absolute counts and percentages of CD3+, CD3 + CD4+, CD3 + CD8+, B and NK cells were determined by single platform technologies. 172 patients received treatment including surgery or chemotherapy after surgery. The factors affecting disease progression were analyzed by Binary Logistic regression. Progression free survival (PFS) calculating survivals were with the method of Kaplan-Meier. The log-rank test and cox's proportional hazard regression (enter method) were used for univariable and multivariable analyses respectively.ResultsRelative to normal controls, patients with NSCLC at different stages showed decreased absolute lymphocyte count obviously, rather than lymphocyte percentages.Different treatments had unlike influence on the homeostasis of lymphocytes and the effects last for a long time. Logistic regression showed CD3 + CD4+ and CD3 + CD8+ could contribute to favorable prognosis. Multivariate analysis of prognostic factors of PFS showed CD3 + CD4+ cell was independent factor for predicting PFS.ConclusionsThe absolute count of CD3+, CD3 + CD4+, CD3 + CD8+, B and NK cells were better indication of the patient's immune state than percentages of each lymphocyte subsets. Immune function was impaired in patients with non-small cell lung. The high level of baseline absolute CD3 + CD4+ cells count contributed to longer progression free survival.Chinese Clinic Trial Registry number: ChiCTR-IOR-17014139; Registry date: 2017/12/25.

Enumeration of lymphocyte subsets during follow-up in the pulmonary tuberculosis patients with co morbid diabetes mellitus

The present study was designed to evaluate percentage of lymphocyte subsets in peripheral blood mononuclear cells of pulmonary tuberculosis patients with diabetes mellitus (TBDM) and household contacts (HHC) at the time of diagnosis and at different intervals of follow-up. T-lymphocyte subsets, monocytes and natural killer cells were evaluated using fluorescence associated cell sorting (FACS) in a total of 125 subjects including TBDM, pulmonary tuberculosis (PTB) patients, HHC, diabetes mellitus (DM) patients and healthy controls (HC), 25 in each category. CD4 proportion was significantly low in TBDM (p = 0.003), PTB (p = 0.0008) and HHC (p = 0.005) when compared to HCs and increased with treatment in PTB at 6 M and 12 M (p = 0.008). CD8 percentage was significantly low in DM (p = 0.01); Significantly high mean percentage was observed with respect to CD14 in TBDM (p = 0.008), PTB (p = 0.018), HHC (p = 0.008) and DM (p = 0.014); with CD16 in TBDM (p = 0.0001), PTB (p = 0.0001), HHC (p = 0.045); with CD56 in TBDM (p = 0.0003), PTB (p = 0.002) and HHC (p = 0.015) respectively when compared to HCs. These results indicate that TBDM patients have altered lymphocyte homeostasis and FACS analysis might have the potential of a non-invasive clinical indicator for the early detection and monitoring of TB in HHC.

Characterization of lamprey (Lampetra japonica) tnfr10-like gene: A potential granulocyte marker molecule and its immune functions

Tumor necrosis factor receptor superfamily (TNFRSF) is an ancient protein superfamily. By binding to tumor necrosis factor (TNF), it can participate in inflammatory response, apoptosis, lymphocyte homeostasis and tissue development. Seven TNFR members have previously been identified in lampreys but detailed functions of TNFR members are not yet to be resolved. Here, we demonstrate some of the distinguishing features of TNFR10-like member which belongs to TNFRSF. The immunohistochemical results indicate that the TNFR10-like protein is abundant in vascular epithelial cells of the lamprey typhlosole and gills. The expression of tnfr10-like gene has a significantly increased at transcription level after Vibrio anguillarum, Staphylococcus aureus and Poly (I:C) stimulation. Notably, TNFR10-like is specifically expressed in the granulocytes of lamprey peripheral blood and supraneural body. Besides, overexpression tnfr10-like gene in HEK-293 T cells cause a decrease in cell activity and able to activate nuclear transcription factor-κB (NF-κB). Together, these results imply that L-TNFR10-like may play a vital role as a potential marker in lamprey granulocytes and may also be involved in regulation of immune response mediated by itself.

Controlled Release of Stem Cell Secretome Attenuates Inflammatory Response against Implanted Biomaterials.

Inflammatory response against implanted biomaterials impairs their functional integration and induces medical complications in the host's body. To suppress such immune responses, one approach is the administration of multiple drugs to halt inflammatory pathways. This challenges patient's adherence and can cause additional complications such as infection. Alternatively, biologics that regulate multiple inflammatory pathways are attractive agents in addressing the implants immune complications. Secretome of mesenchymal stromal cells (MSCs) is a multipotent biologic, regulating the homeostasis of lymphocytes and leukocytes. Here, it is reported that alginate microcapsules loaded with processed conditioned media (pCM-Alg) reduces the infiltration and/or expression of CD68+ macrophages likely through the controlled release of pCM. In vitro cultures revealed that alginate can dose dependently induce macrophages to secrete TNFα, IL-6, IL-1β, and GM-CSF. Addition of pCM to the cultures attenuates the secretion of TNFα (p = 0.023) and IL-6 (p <0.0001) by alginate or lipopolysaccharide (LPS) stimulations. Mechanistically, pCM suppressed the NfκB pathway activation of macrophages in response to LPS (p <0.0001) in vitro and cathepsin activity (p = 0.005) in response to alginate in vivo. These observations suggest the efficacy of using MSC-derived secretome to prevent or delay the host rejection of implants.

Role of autophagy in the pathogenesis of rheumatoid arthritis: Latest evidence and therapeutic approaches

Autophagy is considered as an important intracellular mechanism that degrades cytoplasmic components to furnish additional energy. It has cytoprotective effects through the degradation of intracellular pathogens, damaged organelles, and protein aggregates. On the other hand, there are reports of an association between autophagy and autoimmune diseases. Indeed, it has been evident that autophagy is dysregulated in various autoimmune diseases including rheumatoid arthritis (RA). Autophagy is implicated in the maturation survival and proliferation of various immune and non-immune cells, which play pivotal roles in RA pathogenesis. Additionally, autophagy seems to be involved in citrullination and presentation of citrullinated peptides to T lymphocyte cells. Presentation of citrullinated peptides through MHC compartments to the T cells leads to immune response and chronic inflammation. Evidence suggests that autophagy could be implicated in apoptosis resistance of RA fibroblast-like synoviocyte (RA FLS), osteoclastogenesis, and finally severe bone and cartilage destruction. Since autophagy could be an important phenomenon in RA pathogenesis, we summarized the roles of autophagy in citrullination, osteoclastogenesis, RA FLS cells survival, apoptosis resistance of cells, lymphocyte homeostasis and its clinical outcomes in RA disease.

JNK1 and ERK1/2 modulate lymphocyte homeostasis via BIM and DRP1 upon AICD induction

The Activation-Induced Cell Death (AICD) is a stimulation-dependent form of apoptosis used by the organism to shutdown T-cell response once the source of inflammation has been eliminated, while allowing the generation of immune memory. AICD is thought to progress through the activation of the extrinsic Fas/FasL pathway of cell death, leading to cytochrome-C release through caspase-8 and Bid activation. We recently described that, early upon AICD induction, mitochondria undergo structural alterations, which are required to promote cytochrome-C release and execute cell death. Here, we found that such alterations do not depend on the Fas/FasL pathway, which is instead only lately activated to amplify the cell death cascade. Instead, such alterations are primarily dependent on the MAPK proteins JNK1 and ERK1/2, which, in turn, regulate the activity of the pro-fission protein Drp1 and the pro-apoptotic factor Bim. The latter regulates cristae disassembly and cooperate with Drp1 to mediate the Mitochondrial Outer Membrane Permeabilization (MOMP), leading to cytochrome-C release. Interestingly, we found that Bim is also downregulated in T-cell Acute Lymphoblastic Leukemia (T-ALL) cells, this alteration favouring their escape from AICD-mediated control.

Chlorpyrifos induces apoptosis and autophagy in common carp lymphocytes by influencing the TCR γ-dependent PI3K/AKT/JNK pathway

Chlorpyrifos is an insecticide that is widely used in agricultural production. However, little is known about how chlorpyrifos disrupts lymphocyte homeostasis in common carp. Herein, we identified TCRγ through the results of transcriptome analysis. Subsequently, we established TCR γ knockdown and overexpression models in carp head kidney lymphocyte respectively using RNA interference and the pcDNA3.1 plasmid, respectively. Real-time PCR, fluorescent staining, ultrastructure observation and flow cytometry were used to detect the levels of the PI3K/AKT pathway, autophagy and apoptosis. Our results demonstrated that chlorpyrifos significantly decreased the expression of TCR γ, TCR γ suppression thereby induced increased mRNA expression of TNF-α, Bax, caspase-3, caspase-8, caspase-9 and significantly inhibited the expression of Bcl-2, which indicated that apoptosis was triggered. This conclusion was supported by our flow cytometry and ultrastructure observation results. In addition, the control and TCR γ overexpression groups had normal cell morphology. Moreover, TCR γ suppression activated the expression of Becline-1, ATG5, ATG10, ATG12, ATG16 and reduced the expression of mTOR, with the opposite results observed in the TCR γ overexpression group. Together, these results suggested that TCR γ imbalance triggers apoptosis and autophagy in lymphocyte. Moreover, we found that TCR γ knockdown significantly increased the mRNA expression of JNK and decreased the expression of PI3K and AKT, which indicated that the PI3K/AKT/JNK pathway was activated. Our results reported here indicated that chlorpyrifos induces apoptosis and autophagy in head kidney lymphocyte through the inhibition of TCR γ.

Phosphorylation Modulates Survivin Function in Behcet's Disease.

Purpose: Survivin is critical for proliferation, maturation, homeostasis and differentiation of effector and memory lymphocytes. In this study the baculoviral inhibitors of apoptosis proteins (IAPs) repeat containing 5 (BIRC5) mRNA, survivin, and phosphorylated survivin expression were evaluated in peripheral blood mononuclear cells (PBMCs), and plasma of patients with Behcet’s disease (BD). Methods: In this study, 26 Iranian Azari patients diagnosed with BD and 30 healthy controls were recruited. Total RNA was extracted from PBMCs. The expression level of survivin was measured by quantitative real-time polymerase chain reaction (PCR). Survivin plasma levels were measured using survivin Enzyme-linked immunosorbent assays. Also, western blotting analysis was performed to measure phosphorylated-survivin and survivin levels in PBMCs and plasma of patients with BD. Results: In a pilot study, we showed that BIRC5 gene expression increased in BD patients compared with healthy controls (P<0.05). Western blotting analysis indicated that there was an increase in phosphorylated survivin expression in PBMCs of BD patients. Our data from western blot analysis showed survivin level in plasma samples of BD patients was similar to healthy controls. No significant differences were observed between plasma survivin levels in the BD patients compared with control group (P>0.05). The expression of phosphorylated survivin at Thr34 in PBMCs of BD patients with active disease was increased. Plasma phosphorylated survivin levels in having BD patients were also downregulated compared to healthy individuals. Conclusion: Analysis of PBMCs indicated increasing expression level of phosphorylated survivin in PBMCs of BD patients. There was also a downregulation in phosphorylated survivin levels in plasma of BD patients.

C-MAF, a Swiss Army Knife for Tolerance in Lymphocytes.

Beyond its well-admitted role in development and organogenesis, it is now clear that the transcription factor c-Maf has owned its place in the realm of immune-related transcription factors. Formerly introduced solely as a Th2 transcription factor, the role attributed to c-Maf has gradually broadened over the years and has extended to most, if not all, known immune cell types. The influence of c-Maf is particularly prominent among T cell subsets, where c-Maf regulates the differentiation as well as the function of multiple subsets of CD4 and CD8 T cells, lending it a crucial position in adaptive immunity and anti-tumoral responsiveness. Recent research has also revealed the role of c-Maf in controlling Th17 responses in the intestine, positioning it as an essential factor in intestinal homeostasis. This review aims to present and discuss the recent advances highlighting the particular role played by c-Maf in T lymphocyte differentiation, function, and homeostasis.

Triphenyltin disrupts intracellular Zn2+ homeostasis in rat thymic lymphocytes

Triphenyltin (TPT), previously used as an agricultural fungicide and industrial antifoulant, is now considered an environmental pollutant. The effect of TPT on human health is concerning due to its presence as a contaminant in seafood. In this study, the changes in intracellular Zn2+ concentration ([Zn2+]i) and cellular content of nonprotein thiols ([NPT]i) induced by triphenyltin chloride (TPTCH), were measured in rat thymic lymphocytes. This was studied by flow-cytometry using the fluorescent probes FluoZin-3-AM and 5-chloromethylfluorescein diacetate (5-CMF-DA). Incubation with TPTCH, at 0.1 μM or more (up to 3 μM), increased [Zn2+]i in a concentration-dependent manner. The TPTCH-induced elevation in [Zn2+]i was due to the increase in membrane Zn2+ permeability and intracellular Zn2+ release. Incubation with TPTCH at 0.3 μM significantly increased [NPT]i levels, whereas the addition of an intracellular Zn2+ chelator had no effect on the same. TPT at higher concentrations (1 or 3 μM) reduced [NPT]i. TPT may disturb intracellular Zn2+ signaling in lymphocytes that disturbs cellular functions.

P060 The pharmacologic inhibition of store-operated calcium entry pathway in inflammatory bowel disease

Abstract Background Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition. Results Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.

Molecular evolution of the tnfr gene family and expression profiles in response to pathogens in lamprey(Lethenteron reissneri)

Tumor necrosis factor receptor superfamilies (TNFRSF) are one of essential cytokines and can trigger inflammation, apoptosis, participating lymphocyte homeostasis and tissue development in vertebrates. To gain insights into the evolution and characterization of tnfr genes in lamprey, a jawless vertebrate, we performed a genome-wide and transcriptome survey and identified 7 tnfr genes in the lamprey (Lethenteron reissneri) database. Based on the molecular phylogenetic analysis, 7 L-tnfr genes are divided into three different clusters, and multiple members of tnfr genes family have appeared in lamprey. Meanwhile, protein domains and motifs analysis reveals that TNFRSF are conserved and have typical cysteine-rich domains (CRDs). Synteny results indicates that the L-tnfr neighborhood genes have taken place great changes compared to jawed vertebrates. Real-time quantitative results demonstrate that tnfr gene family plays an important role in the immune defense. This study has a new understanding for origin and evolution of the tnfr gene family in different vertebrates.

YAP and TAZ in Fibroblastic Reticular Cells Support Hematopoiesis and Retention of Lymphocytes in Lymph Nodes

INTRODUCTION: Fibroblastic reticular cells (FRCs) are essential for adaptive immune response and maintaining lymph node (LN) homeostasis. FRCs regulate immune cell entry into the LN and confer compartmentalization of lymphocytes by secretion of essential chemokines. Indeed, FRCs are activated and display an undifferentiated phenotype in LN of patients with follicular lymphoma, but their meaning and mode action are unclear. The mammalian Hippo signaling pathway is an essential regulator of cell fate, differentiation, and homeostasis. Its final downstream effectors YAP and TAZ (YAP/TAZ) are inhibited by LATS1 and LATS2 (LATS1/2) in a phosphorylation-dependent manner. The roles of YAP/TAZ in hematopoiesis are considered to be dispensable under healthy physiological condition, but their significance appears to be context dependent in pathologies including hematologic malignancies. OBJECTIVES: We hypothesized that the Hippo pathway plays important roles in regulating FRCs by interacting with lymphocytes, with relevance to structural and functional properties of LNs. Using FRC-specific YAP/TAZ depletion or hyperactivation mouse models, we sought to investigate the roles of YAP/TAZ in orchestrating lymphocyte homeostasis by FRCs. METHODS: Animal care and experimental procedures were performed under the approval of the Institutional Animal Care and Use Committee (KA2016-12) of KAIST. Yap/Taz∆FRC or Lats1/2∆FRC mouse were generated by crossing FRC-specific Ccl19-Cre or inducible Pdgfrb-Cre-ERT2 mouse with Yapfl/fl/Tazfl/fl or Lats1fl/fl/Lats2fl/fl mouse, respectively. Cre-ERT2-negative but flox/flox-positive littermates were defined as wild-type mice (WT). In order to induce Cre activity, 2 mg of tamoxifen (Sigma-Aldrich) was dissolved in corn oil and intra-peritoneally injected for four consecutive days. Skin-draining LNs were analyzed with immunofluorescence staining, flow cytometry, or fluorescence activated cell sorting. Chemokine expression was measured from sorted FRCs with qRT-PCR, immunoblotting, or ELISA. APC BrdU flow kit (BD Biosciences) was used for cell cycle analysis. Blood count in mice was measured with hemocytometer (pocH-100iV DiFF, Sysmex). RESULTS: Both YAP/TAZ were highly and equally distributed in nucleus and cytoplasm of mouse LN FRCs, in addition to blood and lymphatic endothelial cells. LNs of Yap/Taz∆FRC mice compared with WT revealed a slight reduction in LN weight and reduced total cell number (~64.9%), decreased proportion (~42.9%), and number (~61.1 %) of FRCs among CD45- stromal cells, which were less proliferative (~65.2%) but had no difference in apoptosis. However, neither proliferation nor apoptosis of immune cells was altered. Analysis of isolated FRCs revealed that mRNA levels of lymphoid chemokines for immune cell trafficking and function were significantly attenuated in Yap/TazΔFRC mice compared with WT. Intriguingly, Lats1/2∆FRC mice looked pale from 1 week after birth. We found lower red blood cell (RBC) count (~16.1%) with decreased concentration of hemoglobin (~25.8%) in Lats1/2∆FRC mice compared with WT, suggesting hypochromic anemia. Moreover, although whole body length and overall organ size were small in Lats1/2∆FRC mice compared with WT at 2 weeks after birth, we could not observe any gross or microscopic abnormalities in major organs including brain, heart, lung, liver, and kidney in Lats1/2∆FRC mice. We further analyzed the primary lymphoid organ thymus and the secondary lymphoid organ spleen, as they are both supported by CCL19+ FRCs. Similar to major organs, the overall size of thymus and spleen was smaller in Lats1/2∆FRC mice compared with WT at 2 weeks after birth. However, microscopic analysis of thymus revealed that the border between the cortex and medulla was disrupted by the expanded medulla at the periphery that blends into the surrounding cortex in Lats1/2∆FRC mice, and analysis of spleen also revealed unclear lymphoid follicles with significantly reduced number of immune cells within the expanded white pulp in Lats1/2∆FRC mice compared with WT. CONCLUSIONS: Our findings present YAP/TAZ as essential governors of FRCs by supporting hematopoiesis and lymphocyte homeostasis. Considering that YAP/TAZ are enriched in bone marrow stromal cells, additional work to elucidate their role in promoting bone marrow microenvironment will improve our understanding on hematopoietic stem cell niche. Disclosures No relevant conflicts of interest to declare.

Viruses RIG up intestinal immunity.

The virome, increasingly recognized as a critical component of the mammalian microbiota, modulates host physiology. An antiviral treatment approach reveals that, via RIG-I signaling, the commensal virome is essential for the homeostasis of intestinal intraepithelial lymphocytes.

Commensal viruses maintain intestinal intraepithelial lymphocytes via noncanonical RIG-I signaling.

Much attention has focused on commensal bacteria in health and disease, but the role of commensal viruses is understudied. Although metagenomic analysis shows that the intestine of healthy humans and animals harbors various commensal viruses and the dysbiosis of these viruses can be associated with inflammatory diseases, there is still a lack of causal data and underlying mechanisms to understand the physiological role of commensal viruses in intestinal homeostasis. In the present study, we show that commensal viruses are essential for the homeostasis of intestinal intraepithelial lymphocytes (IELs). Mechanistically, the cytosolic viral RNA-sensing receptor RIG-I in antigen-presenting cells can recognize commensal viruses and maintain IELs via a type I interferon-independent, but MAVS-IRF1-IL-15 axis-dependent, manner. The recovery of IELs by interleukin-15 administration reverses the susceptibility of commensal virus-depleted mice to dextran sulfate sodium-induced colitis. Collectively, our results indicate that commensal viruses maintain the IELs and consequently sustain intestinal homeostasis via noncanonical RIG-I signaling.