GM‐CSF suppresses antioxidant signaling and drives IL‐1β secretion through NRF2 downregulation

GM‐CSF is a potent inflammatory cytokine regulating myeloid cell differentiation, hematopoiesis, and various other functions. It is functionally associated with a number of inflammatory pathologies including rheumatoid arthritis and inflammatory bowel disease. GM‐CSF has been found to promote NLRP3‐dependent IL‐1β secretion, which may have a significant role in driving inflammatory pathologies. However, the molecular mechanisms remain unknown. Here, we show that GM‐CSF induces IL‐1β secretion through a ROS‐dependent pathway. TNF is required for reactive oxygen species (ROS) generation that strikingly does not promote NLRP3 activation, but instead drives ubiquitylation of IL‐1β, promoting its cleavage through basal NRLP3 activity. GM‐CSF regulates this pathway through suppression of antioxidant responses via preventing upregulation of NRF2. Thus, the pro‐inflammatory effect of GM‐CSF on IL‐1β is through suppression of antioxidant responses, which leads to ubiquitylation of IL‐1β and enhanced processing. This study highlights the role of metabolic regulation of inflammatory signaling and reveals a novel mechanism for GM‐CSF to promote inflammation.     

IFN‐γ+IL‐17+Th17 cells regulate fibrosis through secreting IL‐21 in systemic scleroderma

This study aimed to explore the function of IFN‐γ⁺IL‐17⁺Th17 cells on fibrosis in systemic scleroderma (SSc). Blood and skin samples were collected from 20 SSc cases and 10 healthy individuals. The percentage of IFN‐γ⁺IL‐17⁺Th17 cells was detected using flow cytometry. The in vitro induction of IFN‐γ⁺IL‐17⁺Th17 cells was performed adopting PHA and rIL‐12. Gene expression was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR), whereas western blot analysis was adopted for protein analysis. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with SSc stages (P = .031), disease duration (P = .016), activity (P = .025) and skin scores (P < .001). In vitro, IFN‐γ⁺IL‐17⁺Th17 cells could promote the expressions of α‐SMA and COL1A1, revealing increased fibroblasts’ proliferation and enhanced collagen‐secreting capacity. In addition, IL‐21 expression was significantly increased in co‐culture medium of IFN‐γ⁺IL‐17⁺Th17 cells and fibroblasts (P < .001). IL‐21 neutralizer treatment resulted in the down‐regulation of α‐SMA and COL1A1. IL‐21 was confirmed as an effector of IFN‐γ⁺IL‐17⁺Th17 cells in fibrosis process. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with disease activity. IFN‐γ⁺IL‐17⁺Th17 cells could promote fibroblast proliferation and enhance collagen‐secreting ability via producing IL‐21, thus contributing to fibrosis in SSc.     

p38α‐MAPK‐deficient myeloid cells ameliorate symptoms and pathology of APP‐transgenic Alzheimer's disease mice

Alzheimer''s disease (AD), the most common cause of dementia in the elderly, is pathologically characterized by extracellular deposition of amyloid‐β peptides (Aβ) and microglia‐dominated inflammatory activation in the brain. p38α‐MAPK is activated in both neurons and microglia. How p38α‐MAPK in microglia contributes to AD pathogenesis remains unclear. In this study, we conditionally knocked out p38α‐MAPK in all myeloid cells or specifically in microglia of APP‐transgenic mice, and examined animals for AD‐associated pathologies (i.e., cognitive deficits, Aβ pathology, and neuroinflammation) and individual microglia for their inflammatory activation and Aβ internalization at different disease stages (e.g., at 4 and 9 months of age). Our experiments showed that p38α‐MAPK‐deficient myeloid cells were more effective than p38α‐MAPK‐deficient microglia in reducing cerebral Aβ and neuronal impairment in APP‐transgenic mice. Deficiency of p38α‐MAPK in myeloid cells inhibited inflammatory activation of individual microglia at 4 months but enhanced it at 9 months. Inflammatory activation promoted microglial internalization of Aβ. Interestingly, p38α‐MAPK‐deficient myeloid cells reduced IL‐17a‐expressing CD4‐positive lymphocytes in 9 but not 4‐month‐old APP‐transgenic mice. By cross‐breeding APP‐transgenic mice with Il‐17a‐knockout mice, we observed that IL‐17a deficiency potentially activated microglia and reduced Aβ deposition in the brain as shown in 9‐month‐old myeloid p38α‐MAPK‐deficient AD mice. Thus, p38α‐MAPK deficiency in all myeloid cells, but not only in microglia, prevents AD progression. IL‐17a‐expressing lymphocytes may partially mediate the pathogenic role of p38α‐MAPK in peripheral myeloid cells. Our study supports p38α‐MAPK as a therapeutic target for AD patients.     

MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti‐tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL‐2 or IL‐15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA‐181a as a key modulator of human γδ T cell differentiation. We observe that miR‐181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR‐181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR‐181a overexpression restricts their levels of NKG2D and TNF‐α. Upon in silico analysis, we identify two miR‐181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR‐181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next‐generation immunotherapies.     

SARS‐CoV‐2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage

SARS‐CoV‐2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS‐CoV‐2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS‐CoV‐2 viral proteins. Here, we show that the nucleocapsid of SARS‐CoV‐2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS‐CoV‐2‐infected monocytes show enhanced cellular interleukin‐1β (IL‐1β) expression, but reduced IL‐1β secretion. While SARS‐CoV‐2 infection promotes activation of the NLRP3 inflammasome and caspase‐1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS‐CoV‐2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase‐1. These insights into how SARS‐CoV‐2 antagonizes cellular inflammatory responses may open new avenues for treating COVID‐19 in the future.     

Pirfenidone modulates macrophage polarization and ameliorates radiation‐induced lung fibrosis by inhibiting the TGF‐β1/Smad3 pathway

Radiation‐induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation‐induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)‐approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X‐ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor‐β1 (TGF‐β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation‐induced expression of TGF‐β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL‐4–induced M2 macrophage polarization and IL‐13–induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase‐1 (ARG‐1), chitinase 3‐like 3 (YM‐1) and TGF‐β1. Notably, the PFD‐induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa‐B (NF‐κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation‐induced chemokine secretion in MLE‐12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF‐β1 from M2 macrophages by attenuating the activation of TGF‐β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF‐β1/Smad3.     

METTL3 promotes IL‐1β–induced degeneration of endplate chondrocytes by driving m6A‐dependent maturation of miR‐126‐5p

METTL3 is an important regulatory molecule in the process of RNA biosynthesis. It mainly regulates mRNA translation, alternative splicing and microRNA maturation by mediating m6A‐dependent methylation. Interleukin 1β (IL‐1β) is an important inducer of cartilage degeneration that can induce an inflammatory cascade reaction in chondrocytes and inhibit the normal biological function of cells. However, it is unclear whether IL‐1β is related to METTL3 expression or plays a regulatory role in endplate cartilage degeneration. In this study, we found that the expression level of METTL3 and methylation level of m6A in human endplate cartilage with different degrees of degeneration were significantly different, indicating that the methylation modification of m6A mediated by METTL3 was closely related to the degeneration of human endplate cartilage. Next, through a series of functional experiments, we found that miR‐126‐5p can play a significant role in IL‐1β–induced degeneration of endplate chondrocytes. Moreover, we found that miR‐126‐5p can inhibit the PI3K/Akt signalling pathway by targeting PIK3R2 gene, leading to the disorder of cell vitality and functional metabolism. To further determine whether METTL3 could regulate miR‐126‐5p maturation, we first confirmed that METTL3 can bind the key protein underlying pri‐miRNA processing, DGCR8. Additionally, when METTL3 expression was inhibited, the miR‐126‐5p maturation process was blocked. Therefore, we hypothesized that METTL3 can promote cleavage of pri‐miR‐126‐5p and form mature miR‐126‐5p by combining with DGCR8.     

E3 ubiquitin ligase NEDD4L negatively regulates inflammation by promoting ubiquitination of MEKK2

Aberrant activation of inflammation signaling triggered by tumor necrosis factor α (TNF‐α), interleukin‐1 (IL‐1), and interleukin‐17 (IL‐17) is associated with immunopathology. Here, we identify neural precursor cells expressed developmentally down‐regulated gene 4‐like (NEDD4L), a HECT type E3 ligase, as a common negative regulator of signaling induced by TNF‐α, IL‐1, and IL‐17. NEDD4L modulates the degradation of mitogen‐activated protein kinase kinase kinase 2 (MEKK2) via constitutively and directly binding to MEKK2 and promotes its poly‐ubiquitination. In interleukin‐17 receptor (IL‐17R) signaling, Nedd4l knockdown or deficiency enhances IL‐17‐induced p38 and NF‐κB activation and the production of proinflammatory cytokines and chemokines in a MEKK2‐dependent manner. We further show that IL‐17‐induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF‐κB activation but also for NEDD4L‐mediated MEKK2 degradation and the subsequent shutdown of IL‐17R signaling. Importantly, Nedd4l‐deficient mice show increased susceptibility to IL‐17‐induced inflammation and aggravated symptoms of experimental autoimmune encephalomyelitis (EAE) in IL‐17R signaling‐dependent manner. These data suggest that NEDD4L acts as an inhibitor of IL‐17R signaling, which ameliorates the pathogenesis of IL‐17‐mediated autoimmune diseases.     

TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

Nur77 Prevents Osteoporosis by Inhibiting the NF‐κB Signalling Pathway and Osteoclast Differentiation

Inflammation is a major risk factor for osteoporosis, and reducing inflammatory levels is important for the prevention of osteoporosis. Although nuclear receptor 77 (Nur77) protects against inflammation in a variety of diseases, its role in osteoporosis is unknown. Therefore, the main purpose of this study was to investigate the osteoprotective and anti‐inflammatory effects of Nur77. The microCT and haematoxylin and eosin staining results indicated that knockout of Nur77 accelerated femoral bone loss in mice. The enzyme‐linked immunosorbent assay (ELISA) results showed that knockout of Nur77 increased the serum levels of hsCRP and IL‐6. The expression levels of NF‐κB, IL‐6, TNF‐α and osteoclastogenesis factors (TRAP, NFATC1, Car2, Ctsk) in the femurs of Nur77 knockout mice were increased significantly. Furthermore, in vitro, shNur77 promoted the differentiation of RAW264.7 cells into osteoclasts by activating NF‐κB, which was confirmed by PDTC treatment. Mechanistically, Nur77 inhibited osteoclast differentiation by inducing IκB‐α and suppressing IKK‐β. In RAW264.7 cells, overexpression of Nur77 alleviated inflammation induced by siIκB‐α, while siIKK‐β alleviated inflammation induced by shNur77. Consistent with the in vivo studies, we found that compared with control group, older adults with high serum hsCRP levels were more likely to suffer from osteoporosis (OR = 1.76, p < 0.001). Our data suggest that Nur77 suppresses osteoclast differentiation by inhibiting the NF‐κB signalling pathway, strongly supporting the notion that Nur77 has the potential to prevent and treat osteoporosis.     

MiR‐223‐3p inhibits rTp17‐induced inflammasome activation and pyroptosis by targeting NLRP3

The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA‐223‐3p (miR‐223‐3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR‐223‐3p regulates T pallidum‐induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR‐223‐3p levels in syphilis and control samples were determined. The biological function of miR‐223‐3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum‐infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR‐223‐3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR‐223‐3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)‐induced caspase‐1 activation, resulting in decrease in IL‐1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual‐luciferase assay confirmed that NLRP3 is a direct target of miR‐223‐3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR‐223‐3p on T pallidum‐induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR‐223‐3p and NLRP3, caspase‐1, and IL‐1β, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR‐223‐3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum‐infected endothelial cells, implying that miR‐223‐3p could be a potential target for syphilis patients.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

The cultivation conditions affect the aggregation and functionality of β‐cell lines alone and in coculture with mesenchymal stromal/stem cells

The manufacturing of viable and functional β‐cell spheroids is required for diabetes cell therapy and drug testing. Mesenchymal stromal/stem cells (MSCs) are known to improve β‐cell viability and functionality. We therefore investigated the aggregation behavior of three different β‐cell lines (rat insulinoma‐1 cell line [INS‐1], mouse insulinoma‐6 cell line [MIN6], and a cell line formed by the electrofusion of primary human pancreatic islets and PANC‐1 cells [1.1B4]), two MSC types, and mixtures of β‐cells and MSCs under different conditions. We screened several static systems to produce uniform β‐cell and MSC spheroids, finding cell‐repellent plates the most suitable. The three different β‐cell lines differed in their aggregation behavior, spheroid size, and growth in the same static environment. We found no major differences in spheroid formation between primary MSCs and an immortalized MSC line, although both differed with regard to the aggregation behavior of the β‐cell lines. All spheroids showed a reduced viability due to mass transfer limitations under static conditions. We therefore investigated three dynamic systems (shaking multi‐well plates, spinner flasks, and shaking flasks). In shaking flasks, there were no β‐cell‐line‐dependent differences in aggregation behavior, resulting in uniform and highly viable spheroids. We found that the aggregation behavior of the β‐cell lines changed in a static coculture with MSCs. The β‐cell/MSC coculture conditions must be refined to avoid a rapid segregation into distinct populations under dynamic conditions.     

Decomposing Spatial β‐Diversity in the temperate forests of Northeastern China

β‐Diversity, which describes the extent of change in species composition in a given region, has become a core issue in ecology in recent years. However, it is hard to understand the underlying mechanisms of β‐diversity by using indices that yield identical values under species replacement and nestedness pattern. Partitioning β‐diversity into turnover (caused by species replacement among plots) and nestedness components (caused by species loss or gain among plots) may provide improved understanding of the variation in species composition. Here, we collected presence–absence data of 456 one‐tenth ha circular plots in the temperate forests of Northeastern China spanning a latitudinal range of 12° (41–53°N). We decomposed β‐diversity to assess the relative contribution of the turnover and nestedness components across latitudinal gradients. We used regression analysis to assess the relationship between spatial distance and β‐diversity. We applied variation partitioning to evaluate the importance of the measured environmental and spatial variables in explaining β‐diversity. We used the Tukey honest significant difference test to test the differences of β‐diversity along latitudinal gradients. Pearson correlations (r) and significance (p‐value) were computed using the Mantel tests to verify the relationship between distance and β‐diversity. The ANOVA test was used to verify whether the variation of β‐diversity explained by the environment and distance was significant. Our results showed that (1) β‐diversity and the turnover component were higher at low latitudes (zones A and B) than at high latitudes (zones C and D), while there was no relationship between the nestedness component and latitude. (2) The turnover component was dominant. (3) The spatial distance explained more variation of β‐diversity than the measured environmental factors. Therefore, we conclude that β‐diversity is mainly a product of species turnover in our temperate forests, suggesting that different localities harbor different species. We find that decomposing β‐diversity into the turnover and nestedness components is a useful approach to explore the variation of community composition and their causes.     

Anti‐inflammatory properties of lemon‐derived extracellular vesicles are achieved through the inhibition of ERK/NF‐κB signalling pathways

Chronic inflammation is associated with the occurrence of several diseases. However, the side effects of anti‐inflammatory drugs prompt the identification of new therapeutic strategies. Plant‐derived extracellular vesicles (PDEVs) are gaining increasing interest in the scientific community for their biological properties. We isolated PDEVs from the juice of Citrus limon L. (LEVs) and characterized their flavonoid, limonoid and lipid contents through reversed‐phase high‐performance liquid chromatography coupled to electrospray ionization quadrupole time‐of‐flight mass spectrometry (RP‐HPLC–ESI‐Q‐TOF‐MS). To investigate whether LEVs have a protective role on the inflammatory process, murine and primary human macrophages were pre‐treated with LEVs for 24 h and then were stimulated with lipopolysaccharide (LPS). We found that pre‐treatment with LEVs decreased gene and protein expression of pro‐inflammatory cytokines, such as IL‐6, IL1‐β and TNF‐α, and reduced the nuclear translocation and phosphorylation of NF‐κB in LPS‐stimulated murine macrophages. The inhibition of NF‐κB activation was associated with the reduction in ERK1‐2 phosphorylation. Furthermore, the ability of LEVs to decrease pro‐inflammatory cytokines and increase anti‐inflammatory molecules was confirmed ex vivo in human primary T lymphocytes. In conclusion, we demonstrated that LEVs exert anti‐inflammatory effects both in vitro and ex vivo by inhibiting the ERK1‐2/NF‐κB signalling pathway.     

Fungal‐induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL‐22

Incorporation of microbiome data has recently become important for prevention, diagnosis, and treatment of colorectal cancer, and several species of bacteria were shown to be associated with carcinogenesis. However, the role of commensal fungi in colon cancer remains poorly understood. Here, we report that mice lacking the c‐type lectin Dectin‐3 (Dectin‐3 ^−/−) show increased tumorigenesis and Candida albicans burden upon chemical induction. Elevated C. albicans load triggered glycolysis in macrophages and interleukin‐7 (IL‐7) secretion. IL‐7 induced IL‐22 production in RORγt⁺ (group 3) innate lymphoid cells (ILC3s) via aryl hydrocarbon receptor and STAT3. Consistently, IL‐22 frequency in tumor tissues of colon cancer patients positively correlated with fungal burden, indicating the relevance of this regulatory axis in human disease. These results establish a C. albicans‐driven crosstalk between macrophages and innate lymphoid cells in the intestine and expand our understanding on how commensal mycobiota regulate host immunity and promote tumorigenesis.