IL-10 KO Mice Research Articles

Frontline Science: IL-18 primes murine NK cells for proliferation by promoting protein synthesis, survival, and autophagy.

Combined stimulation by IL-2 and IL-18 effectively promotes proliferation of NK cells, whereas singular stimulation does not. In this study, synergistic effects of these cytokines on NK cells proliferation was analyzed, focusing on the roles of IL-18. In splenic resting NK cells from IL-18KO mice, IL-18 rapidly activated NF-κB independently of IL-2, and activated or up-regulated various molecules downstream of PI3K/AKT and mTOR, including S6, Bcl-XL, ATG5, and LC3II, accompanying increases in cell growth and survival. Thus, IL-18 alone was revealed to augment various cellular processes (gene transcription, protein synthesis, survival) in the absence or presence of IL-2. Notably, combined IL-18 and IL-2 promoted autophagosome formation. In addition, priming NK cells with IL-18 augmented IL-2R, especially CD25, and enabled cells to respond to IL-2, resulting inactivation of STAT3 and STAT5,followed by increase of cyclin B1 leading to proliferation. However, IL-2 alone failed to activate STAT3 or STAT5 in resting IL18KO NK cells. These results clarify the distinct roles of IL-2 and IL-18 in NK cell proliferation, and the intrinsic roles of IL-18 in various cellular processes, suggesting a range of functions of IL-18 expressed in an array of nonhematopoietic cells.

Interleukin-10 produced by myeloid-derived suppressor cells is critical for the induction of Tregs and attenuation of rheumatoid inflammation in mice

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid progenitor cells with immunoregulatory function. MDSCs play critical roles in controlling the processes of autoimmunity but their roles in rheumatoid arthritis (RA) are controversial. The present study was undertaken to investigate whether MDSCs have therapeutic impact in mice with collagen-induced arthritis (CIA), an animal model of RA. We also examined the mechanisms underlying the anti-arthritic effect of MDSCs. In vitro treatment with MDSCs repressed IL-17 but increased FOXP3 in CD4+ T cells in mice. In vivo infusion of MDSCs markedly ameliorated inflammatory arthritis. Th17 cells and Th1 cells were decreased while Tregs were increased in the spleens of MDSCs-treated mice. MDSCs profoundly inhibited T cell proliferation. Addition of anti-IL-10 almost completely blocked the anti-proliferative effects of MDSCs on T cells. Anti-IL-10 blocked the expansion of Tregs by MDSCs. However, infusion of MDSCs from IL-10 KO mice failed to suppress inflammatory arthritis. MDSCs could reciprocally regulate Th17/Treg cells and suppress CIA via IL-10, suggesting that MDSCs might be a promising therapeutic strategy for T cell mediated autoimmune diseases including RA.

P025 CYSTATHIONINE-GAMMA-LYASE REGULATES MACROPHAGE INTERLEUKIN-10 (IL-10) PRODUCTION IN COLITIS

Cystathionine-β-synthase (CBS) & cystathionine-γ-lyase (CSE), convert homocysteine into cysteine & produce H2S, an anti-inflammatory mediator. In rodent colitis models, CBS & CSE activity is elevated and inhibition or knockout of CSE worsens colitis & decreases levels of anti-inflammatory cytokine IL-10. IL-10 KO mice develop spontaneous colitis accompanied by homocysteinemia & decreased H2S production, which are reversed by IL-10 administration. However, the cellular source of CSE-dependent IL-10 production is unknown. We hypothesize that macrophage-derived CSE is critical for IL-10 production. Human colitis & normal colon specimens were collected. CSE knockout (CSE KO) mice and wild-type (WT) controls received either 7 days of colitis-inducer DSS (2% in drinking water) or vehicle. CSE & cell-type specific markers were evaluated in human/mouse colons by immunofluorescent microscopy & flow cytometry. Peritoneal macrophages from CSE KO & WT mice (N = 6) were isolated & treated with CSE inhibitor propargyl glycine (PAG), H2S donor NaHS or vehicle. IL-10 & β-actin expression levels were determined by qRT-PCR. Confocal microscopy of colitis & normal human colon tissue localized CSE to cells of the lamina propria expressing CD45, a marker of hematopoietic cells. Colonic tissue from mice receiving either DSS or vehicle also showed CSE staining in the lamina propria co-localized with macrophage marker F4/80. Flow cytometry showed co-expression of CSE with F4/80 but not Treg marker FOXP3. Comparison of macrophages from CSE KO to WT mice revealed a 6-fold reduction of IL-10 mRNA. Further, treatment of macrophages from WT mice with PAG inhibited IL-10 expression compared to controls. Addition of NaHS did not alter CSE KO macrophage expression of IL-10. We demonstrate that in macrophages CSE & IL-10 expression are linked in an H2S-independent manner. This suggests that CSE regulates inflammation through both H2S & IL-10.

P070 MACROPHAGE-EXPRESSED ERBB4 PLAYS A PROTECTIVE ROLE IN THE ONSET AND RESOLUTION OF EXPERIMENTAL COLITIS

Pro-inflammatory macrophages (Mø) are crucial for onset of intestinal inflammation, and defects that enable persistence of these cells may contribute to inflammatory bowel disease. We recently showed that the ErbB4 receptor tyrosine kinase is induced on Mø by pro-inflammatory activation, and that subsequent stimulation with the ErbB4-specific ligand neuregulin-4 kills these cells. This suggests that Mø-expressed ErbB4 limits inflammation, but the cell type specificity of this effect and the role of ErbB4 in regulating colitis onset and recovery in vivo is untested. We hypothesize that selective Mø-expressed ErbB4 limits onset and promotes recovery from experimental colitis. To test the cell type specificity of ErbB4 induction, we generated bone-marrow derived Mø, dendritic cells, and neutrophils from C57BL/6 mice. To test the role of ErbB4 in experimental colitis, mice with a myeloid cell-specific ErbB4 deletion (LysMCre/ErbB4FF) were crossed with IL-10KO mice (LysMCre/ErbB4FF/IL-10KO) or subjected to a DSS colitis recovery model (6 days DSS + 6 days water). The induction (ten-fold, p<0.01) of ErbB4 seen in pro-inflammatory Mø was not observed in activated dendritic cells or neutrophils. LysMCre/ErbB4FF/IL-10KO displayed inflammation markers (elevated fecal lipocalin-2 and colonic IL-17) by 8 weeks of age, when littermate controls were still healthy. LysMCre/ErbB4FF mice had impaired weight recovery, shorter colon lengths, and elevated fecal lipocalin-2 levels (p<0.05 versus littermate controls) 12 days after DSS. ErbB4 induction by challenge in innate immune cells was specific to pro-inflammatory Mø. Loss of Mø ErbB4 signaling accelerated IL-10KO colitis onset and impaired recovery from DSS colitis, suggesting that this receptor normally drives an anti-inflammatory feedback loop that limits colitis onset and promotes resolution.

Gastrointestinal Flora Dictates the Biological Response in Murine Transfusion Related Acute Lung Injury (TRALI)

Transfusion related acute lung injury (TRALI) is a syndrome of respiratory distress which occurs within 6 hours of blood transfusion. It is the leading cause of transfusion-related fatalities and the pathogenesis is incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI. Recently, using novel murine models of TRALI, CD4+T cells were found to be important protector cells against antibody-mediated TRALI and administration of interleukin (IL)-10 was demonstrated to be a successful treatment strategy for TRALI, rescuing mice therapeutically from pulmonary edema, the hallmark of acute lung injury (Kapur et al, Blood 2017, 129(18):2557-2569). Whether the gut microbiome plays any role in the development of TRALI is currently unknown. For that purpose, we compared the biological TRALI response in mice housed in a barrier-free (BF) setting versus mice housed in a specific pathogen-free (SPF) environment. We utilized our TRALI model in which C57BL/6 mice were first depleted of CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes for several parameters including pulmonary edema (lung wet-to-dry weight ratios, W/Ds), rectal temperatures (indicative of systemic shock), plasma levels of macrophage inflammatory protein (MIP)-2 (murine homologue of IL-8, a neutrophil chemoattractant), levels of pulmonary neutrophils (major effector cells in TRALI) and lung histology. We observed that after one week of housing, baseline rectal temperatures of BF mice significantly increased from 38.7o C to 39.5° C (p<0.001) while SPF mice remained stable around 38.3 °C. When comparing BF versus SPF mice, the baseline temperature of BF mice was significantly higher than SPF mice after one week of housing (P<0.0001). The gut flora was subsequently depleted in BF mice via administration of broad-spectrum antibiotics through the drinking water. When both aerobic and anaerobic gut microbes were efficiently depleted after a week of antibiotic treatment, rectal temperatures of the treated BF mice significantly dropped again to 38.7 °C (P<0.05). When inducing TRALI by depleting CD4+ T cells and injecting TRALI-antibodies, the rectal temperatures of the untreated BF mice remained low while the rectal temperatures of SPF mice and gut flora depleted-BF mice normalized again from 60 minutes onwards (34.9 versus 36.8 versus 37.8 °C, respectively). Moreover, the untreated BF mice suffered from TRALI while the SPF mice and the gut flora depleted-BF mice were protected from TRALI development (lung W/Ds 5.77 versus 4.63 versus 4.49, respectively) which was also evident from lung histology-analyses. The prevention of TRALI in SPF mice and gut flora depleted BF mice was paralleled by decreased plasma MIP-2 levels compared to the untreated BF mice which suffered from TRALI (MIP-2: 0.09 versus 0.08 versus 82.94 pg/ml, respectively) which also corresponded to pulmonary neutrophil numbers. As previously shown, low IL-10 levels were associated with TRALI development and IL-10 KO mice were found to be susceptible to antibody-mediated murine TRALI (without prior in vivo cell-depletion). However, when IL-10 KO mice were housed under SPF conditions and injected with TRALI-inducing antibodies, they did not display increased lung W/Ds levels compared to naïve SPF mice indicating that the gut flora precedes and dictates the biological TRALI response. These data collectively link the gut flora to the development of antibody-mediated TRALI in mice and antibiotic treatment prevents TRALI and may perhaps prevent the onset of human TRALI which may help in TRALI risk-assessment prior to transfusion. DisclosuresSemple:Rigel: Consultancy; Novartis: Consultancy; Amgen: Consultancy; UCB: Consultancy.

IL-17 is a protection effector against the adherent-invasive Escherichia coli in murine colitis

Inflammatory bowel disease (IBD) is caused by aberrant immune responses to the gut microbiota. Among the gut microbiota, adherent-invasive Escherichia Coli (AIEC) is thought to be the pathogen through invading the intestinal epithelial cells and causing inflammation. IL-17 secretion increase, induced by enhanced bacterial adhesion to the intestine epithelium, could on one hand protect the mucosa, but on the other hand, over amount of IL-17 initializes inflammation reactions that in turn damages the mucosa. The relationship between IL-17 and AIEC is still unclear. In this study, we tried to elucidate the function of IL-17 in AIEC-mediated colitis. Wild type (WT) and IL-17 knockout (IL-17 KO) mice were inoculated with AIEC strain E. coli LF82 and treated with dextran sodium sulphate (DSS). Histological examination of the colon was performed. Mucosa damage was assessed and scored. IL-22 and IL-17 in colon tissues were detected by ELISA, qPCR and immunohistochemistry methods. Transient AIEC colonization in IL-17 KO mice resulted in increased intestinal epithelial damage, systemic bacterial burden and mortality compared with WT controls. Moreover, IL-17 is required for the induction of IL-22 in the experimental animal models during AIEC strain E. coli LF82 colonization. These results indicate IL-17 plays a protective role in AIEC strain E. coli LF82 induced colitis by promoting IL-22 secretion.

Mice lacking interleukin-18 gene display behavioral changes in animal models of psychiatric disorders: Possible involvement of immunological mechanisms

Preclinical and clinical evidence suggests pro-inflammatory cytokines might play an important role in the neurobiology of schizophrenia and stress-related psychiatric disorders. Interleukin-18 (IL-18) is a member of the IL-1 family of cytokines and it is widely expressed in brain regions involved in emotional regulation. Since IL-18 involvement in the neurobiology of mental illnesses, including schizophrenia, remains unknown, this work aimed at investigating the behavior of IL-18 null mice (KO) in different preclinical models: 1. the prepulse inhibition test (PPI), which provides an operational measure of sensorimotor gating and schizophrenic-like phenotypes; 2. amphetamine-induced hyperlocomotion, a model predictive of antipsychotic activity; 3. resident-intruder test, a model predictive of aggressive behavior. Furthermore, the animals were submitted to models used to assess depressive- and anxiety-like behavior. IL-18KO mice showed impaired baseline PPI response, which was attenuated by d-amphetamine at a dose that did not modify PPI response in wild-type (WT) mice, suggesting a hypodopaminergic prefrontal cortex function in those mice. d-Amphetamine, however, induced hyperlocomotion in IL-18KO mice compared to their WT counterparts, suggesting hyperdopaminergic activity in the midbrain. Moreover, IL-18KO mice presented increased basal levels of IL-1β levels in the hippocampus and TNF-α in the prefrontal cortex, suggesting an overcompensation of IL-18 absence by increased levels of other proinflammatory cytokines. Although no alteration was observed in the forced swimming or in the elevated plus maze tests in naïve IL-18KO mice, these mice presented anxiogenic-like behavior after exposure to repeated forced swimming stress. In conclusion, deletion of the IL-18 gene resembled features similar to symptoms observed in schizophrenia (positive and cognitive symptoms, aggressive behavior), in addition to increased susceptibility to stress. The IL-18KO model, therefore, could provide new insights into how changes in brain immunological homeostasis induce behavioral changes related to psychiatric disorders, such as schizophrenia.

Ablation of IL-17 expression moderates experimental autoimmune myasthenia gravis disease severity

An array of cytokines influences the pathogenesis of early onset myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG). Patients with MG, in particular those with more severe weakness, have elevations of the pro-inflammatory cytokine IL-17 in the blood. We assessed the role of IL-17A in autoimmunity by inducing EAMG in mice with knockout of IL-17 and found a reduction of EAMG severity, but not a complete ablation of disease. The IL-17ko mice had no evidence of weakness, low levels of acetylcholine receptor antibodies, and retention of acetylcholine receptor at the neuromuscular junction. Splenic germinal center size was reduced in EAMG IL-17ko mice along with elevations of Foxp3 and BCL-6 gene expression, suggesting a shift away from pro-inflammatory signals. The results emphasize the importance of IL-17 in EAMG development and that IL-17 independent pathways drive the autoimmune reaction.

Interleukin-18 gene deletion protects against sepsis-induced cardiac dysfunction by inhibiting PP2A activity

BackgroundInterleukin-18 (IL-18) neutralization protects against lipopolysaccharide (LPS)-induced injuries, including myocardial dysfunction. However, the mechanism is yet to be fully elucidated. The aim of the present study was to determine whether IL-18 gene deletion prevents sepsis-induced cardiac dysfunction and to elucidate the potential mechanisms underlying IL-18-mediated cardiotoxicity by LPS. Methods and resultsTen-week-old male wild-type (WT) and IL-18 knockout (IL-18 KO) mice were intraperitoneally administered LPS. Serial echocardiography showed better systolic pump function and less left ventricular (LV) dilatation in LPS-treated IL-18 KO mice compared with those in LPS-treated WT mice. LPS treatment significantly decreased the levels of phospholamban (PLN) and Akt phosphorylation in WT mice compared with those in saline-treated WT mice, while the LPS-induced decrease in the phosphorylation levels was attenuated in IL-18 KO mice compared with that in WT mice. IL-18 gene deletion also attenuated an LPS-induced increase of type 2 protein phosphatase 2A (PP2A) activity, a molecule that dephosphorylates PLN and Akt. There was no difference in type 1 protein phosphatase (PP1) activity. To address whether IL-18 affects PLN and Akt phosphorylation via PP2A activation in cardiomyocytes, rat neonatal cardiac myocytes were cultured and stimulated using 100ng/ml of recombinant rat IL-18. Exogenous IL-18 decreased the level of PLN and Akt phosphorylation in cardiomyocytes. PP2A activity but not PP1 activity was increased by IL-18 stimulation in cardiomyocytes. ConclusionsIL-18 plays a pivotal role in advancing sepsis-induced cardiac dysfunction, and the mechanisms underlying IL-18-mediated cardiotoxicity potentially involve the regulation of PLN and Akt phosphorylation through PP2A activity.

The role of IL-10 and FasL in regulating Th1 and Th17 cell responses toward recoverin in autoimmune retinopathy

Abstract Autoimmune retinopathy (AIR) is a condition that manifests in acute and progressive vision loss in humans. Antibodies against the retinal protein recoverin are frequently detected in AIR patients. We have established a murine model to better understand the immunopathology of AIR. Mice were immunized subcutaneously with recombinant mouse recoverin (100–200 μg) emulsified in complete Freund’s adjuvant (CFA) and intraperitoneally with pertussis toxin (400 ng). After three weeks, the immunized mice were scanned with optical coherence tomography (OCT) for detection of pathological changes in the eyes. Cells from spleen and draining lymph nodes were restimulated with recoverin (5 μg/ml) for 4 days. Immunization of C57BL/6 and BALB/c mice with mouse recoverin resulted in recoverin-specific T cell responses. Production of IFN-γ and IL-17 were observed in cells harvested from draining lymph nodes and spleen of the immunized mice upon restimulation in vitro with recoverin. Immunization also led to production of high titers of recoverin-specific antibodies (1/400) in the immunized mice. The recoverin-specific Th1 and Th17 cell response was augmented in IL-10 KO mice, whereas humoral response was unaffected. More importantly, the number of lymphocytes infiltrated into the eyes dramatically increased in IL-10 KO mice upon immunization with recoverin. Immunization of Fas ligand deficient GLD mice with recoverin antigen led to a predominant Th17 cell response toward recoverin. These new insights regarding the anti-recoverin immune response have led us to hypothesize that skewing of recoverin-specific T cell response toward inflammatory Th1 and/or Th17 responses can drive the pathogenesis of AIR.

Increased NCR negative ILC 3 may be indicative for the early development of spontaneous colitis in the IL 10 knockout mouse

Abstract The IL10 knockout (KO) mouse is a model for environmentally and genetically triggered spontaneous colitis. The response of the adaptive immune system to these changes has been studied extensively in this model. However, the response of the innate immune system, specifically the response of innate lymphoid cell (ILCs) populations to the genetic background of the mouse and the environmental stimulus of introducing microbiota has not been well studied. Herein, we evaluated the response of ILCs and Lin marker positive lymphocytes to changes in genotype (wild type Mice B6 in SPF to IL10 KO B6 mice in SPF) and also compared IL10KO mice who were either germ free or SPF exposed. To accomplish this, IL10 knockout mice were exposed to SPF conditions for three weeks. LPMCs were isolated from the colon and small bowel. Innate lymphoid cells were identified as Lin− CD90.2+CD127+cells, and within total ILC, ILC2s were ST2+ cells, ILC1s were ST2−CD117− cells, and ILC3s were ST2−CD117+NKp46+/−cells. As expected, we saw a less developed adaptive immune response in GF IL10 KO mice, with up regulation in this compartment following SPF exposure (Lin+ population increase 1.87 fold). Upon comparison of WT SPF versus IL10 KO SPF, we observed a 5 fold increase in total ILCs and an 18% increase in NCR-ILC3 in the SPF. Comparison of IL10 KO GF versus SPF revealed a 1.8 fold increase in total ILC and a 3% increase in NCR-ILC3s. In conclusion, we show that both genetic and environmental factors play a role in the innate immune changes that occur prior to the development of spontaneous colitis in the IL10 KO mouse and that the shift towards NCR-ILC 3 maybe an early marker for the development of spontaneous colitis.

Interleukin-15 Enables Septic Shock by Maintaining Natural Killer Cell Integrity and Function

Abstract Interleukin-15 (IL-15) is essential for development, differentiation and function of natural killer (NK) and memory (m) CD8+T cells. Our lab previously showed that NK and CD8+T cells facilitate systemic inflammation and physiological dysfunction during septic shock. However, factors that regulate NK and CD8+T cell function during sepsis are not well characterized. We hypothesize that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+T cell numbers and function. To test our hypothesis, the response of IL-15-deficient (IL-15 KO) mice to sepsis was assessed. IL-15 KO mice showed improved survival, attenuated hypothermia, and less pro-inflammatory cytokine production during septic shock caused by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS). Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15 neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion and conferred protection if given 4 days prior to septic challenge. IL-15 SA treatment amplified septic shock, which was prevented by NK cell or IFNγ depletion. IL-15 SA treatment also exacerbated septic shock caused by CLP if given after the onset of sepsis. In conclusion, endogenous IL-15 doesn’t directly augment the pathogenesis of sepsis but enables septic shock by maintaining NK cell numbers and function. Exogenous IL-15 SA exacerbates severity of sepsis by activating NK cells and facilitating IFNγ production.

Type 1 regulatory T cells (Tr1) homeostasis and function in aging

Abstract Type 1 regulatory T cells (Tr1) are a unique population of CD4+ Foxp3− cells that express high levels of IL-10, and have been defined based on their expression of CD49b and LAG-3. Despite the critical roles played by Tr1 cells in controlling T cell responses in autoimmunity and infection, the mechanisms underlying the homeostasis of Tr1 cells remain unclear. Here, we investigated the homeostasis, phenotype and function of Tr1 cells with age. We found that Tr1 cells accumulated dramatically with age. Further, Tr1 cells produced more IL-10 per cell compared to Foxp3+ Treg. While aged Tr1 cells expressed significant levels of c-Maf and Egr-2, two of the Tr1 defining transcription factors, they largely lacked expression of CD49b and were heterogeneous in LAG-3 and ICOS expression. Despite this heterogeneity, LAG-3 identified a population of aged Tr1 cells that potently inhibited T-cell proliferation in a partially IL-10-dependent manner in vitro and controlled colitis severity in vivo. Additional flow cytometric analysis revealed that roughly half of the IL-10 producing cells co-produced IFN-γ. Further, single cell mRNA-seq analysis revealed that IL-10+IFN-γ− cells had a TGF-beta signature, while IL-10+IFN-γ+ cells had a STAT4 signature. Strikingly, IL-6 was critical for overall Tr1 cell accumulation, as aged IL-6 KO mice showed a profound reduction in Tr1 cells and their expression of c-Maf. Combined, these data show that IL-6 promotes Tr1 accumulation with age and provide a new insight into a novel feedback loop whereby an inflammatory cytokine drives accrual of an anti-inflammatory cell population.

Disrupted Ultradian Activity Rhythms and Differential Expression of Several Clock Genes in Interleukin-6-Deficient Mice.

The characteristics of the cycles of activity and rest stand out among the most intensively investigated aspects of circadian rhythmicity in humans and experimental animals. Alterations in the circadian patterns of activity and rest are strongly linked to cognitive and emotional dysfunctions in severe mental illnesses such as Alzheimer’s disease (AD) and major depression (MDD). The proinflammatory cytokine interleukin 6 (IL-6) has been prominently associated with the pathogenesis of AD and MDD. However, the potential involvement of IL-6 in the modulation of the diurnal rhythms of activity and rest has not been investigated. Here, we set out to study the role of IL-6 in circadian rhythmicity through the characterization of patterns of behavioral locomotor activity in IL-6 knockout (IL-6 KO) mice and wild-type littermate controls. Deletion of IL-6 did not alter the length of the circadian period or the amount of locomotor activity under either light-entrained or free-running conditions. IL-6 KO mice also presented a normal phase shift in response to light exposure at night. However, the temporal architecture of the behavioral rhythmicity throughout the day, as characterized by the quantity of ultradian activity bouts, was significantly impaired under light-entrained and free-running conditions in IL-6 KO. Moreover, the assessment of clock gene expression in the hippocampus, a brain region involved in AD and depression, revealed altered levels of cry1, dec2, and rev-erb-beta in IL-6 KO mice. These data propose that IL-6 participates in the regulation of ultradian activity/rest rhythmicity and clock gene expression in the mammalian brain. Furthermore, we propose IL-6-dependent circadian misalignment as a common pathogenetic principle in some neurodegenerative and neuropsychiatric disorders.

IL-15 Enables Septic Shock by Maintaining NK Cell Integrity and Function.

Interleukin 15 is essential for the development and differentiation of NK and memory CD8+ (mCD8+) T cells. Our laboratory previously showed that NK and CD8+ T lymphocytes facilitate the pathobiology of septic shock. However, factors that regulate NK and CD8+ T lymphocyte functions during sepsis are not well characterized. We hypothesized that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+ T cell integrity. To test our hypothesis, the pathogenesis of sepsis was assessed in IL-15-deficient (IL-15 knockout, KO) mice. IL-15 KO mice showed improved survival, attenuated hypothermia, and less proinflammatory cytokine production during septic shock caused by cecal ligation and puncture or endotoxin-induced shock. Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15-neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion if given 4 d prior to septic challenge and conferred protection. IL-15 SA treatment amplified endotoxin shock, which was prevented by NK cell or IFN-γ depletion. IL-15 SA treatment also exacerbated septic shock caused by cecal ligation and puncture when given after the onset of sepsis. In conclusion, endogenous IL-15 does not directly augment the pathogenesis of sepsis but enables the development of septic shock by maintaining NK cell numbers and integrity. Exogenous IL-15 exacerbates the severity of sepsis by activating NK cells and facilitating IFN-γ production.

Endogenous IL-33 Deficiency Exacerbates Liver Injury and Increases Hepatic Influx of Neutrophils in Acute Murine Viral Hepatitis.

The alarmin IL-33 has been described to be upregulated in human and murine viral hepatitis. However, the role of endogenous IL-33 in viral hepatitis remains obscure. We aimed to decipher its function by infecting IL-33-deficient mice (IL-33 KO) and their wild-type (WT) littermates with pathogenic mouse hepatitis virus (L2-MHV3). The IL-33 KO mice were more sensitive to L2-MHV3 infection exhibiting higher levels of AST/ALT, higher tissue damage, significant weight loss, and earlier death. An increased depletion of B and T lymphocytes, NKT cells, dendritic cells, and macrophages was observed 48 h postinfection (PI) in IL-33 KO mice than that in WT mice. In contrast, a massive influx of neutrophils was observed in IL-33 KO mice at 48 h PI. A transcriptomic study of inflammatory and cell-signaling genes revealed the overexpression of IL-6, TNFα, and several chemokines involved in recruitment/activation of neutrophils (CXCL2, CXCL5, CCL2, and CCL6) at 72 h PI in IL-33 KO mice. However, the IFNγ was strongly induced in WT mice with less profound expression in IL-33 KO mice demonstrating that endogenous IL-33 regulated IFNγ expression during L2-MHV3 hepatitis. In conclusion, we demonstrated that endogenous IL-33 had multifaceted immunoregulatory effect during viral hepatitis via induction of IFNγ, survival effect on immune cells, and infiltration of neutrophils in the liver.

Postnatal changes of interleukin-18 receptor immunoreactivity in neurons of the retrosplenial cortex in wild-type and interleukin-18 knock out mice.

Interleukin-18 (IL-18), which is involved in the inflammatory response, is also found in the cerebral cortex. IL-18 receptor-immunoreactive (IL-18R-ir) neurons are present in layer V of the retrosplenial cortex (RSC). In the adult IL-18 knock out (KO) mice, no IL-18R-ir neurons but many degenerated neurons are present in layer V of the RSC, suggesting that any changes in the neurons of layer V have occurred during postnatal development. We examined changes of IL-18R expression during postnatal development. In the wild-type mice, many IL-18R-ir neurons were present in layers II, III and VI of the RSC in 2-week-old mice, whereas they were sparsely observed in only layer III in 3-week-old mice. No IL-18R-ir neurons were present in 4- and 5-week-old mice. In older than 6-week-old mice, many IL-18R-ir neurons were present in layers V and VI. The IL-18KO mice showed IL-18R-ir neurons in layers II, III and VI at 2-weeks-old, and a few in layer III at 3-week-old mice, similar to that in the wild-type mice. No IL-18R-ir neurons were found in mice older than 4 weeks of age. Thus, IL-18 or IL-18R seem to be involved in the construction of neural circuits corresponding to events after 3-weeks of age.

GPR120, a potential therapeutic target for experimental colitis in IL-10 deficient mice.

It has been proved that interleukin-10-knockout (IL-10 KO) mice display the most similar characteristics to that of human Crohn's disease (CD). Docosahexaenoic acid (DHA) has well established beneficial effects on human and animal models health with potent anti-inflammatory effects with poorly understood mechanisms. This study was aimed at figuring out whether DHA could ameliorate the Crohn's colitis by activating GPR120 and whether GPR120 could be a potential therapeutic target for CD.16 week-old mice included in our present study were divided into three groups, WT group, IL-10 KO group and DHA group(IL-10 KO mice with DHA treatment, i.g., 35.5mg/kg/d), containing 8 mice in each group. The severity of colitis, pro-inflammatory cytokines concentrations, the expression/distribution of protein GPR120 and TAK1/IKK-α/IkB-α/p65 pathway in the proximal colons were evaluated at the end of the experiment. Administration of DHA showed promising results in the experimental chronic colitis (demonstrated by reduced infiltration of inflammatory cells, lowered inflammation scores, decreased pro-inflammatory cytokines) and body weight loss improvement. Moreover, in the DHA-treated mice, enhanced expression and improved distribution integrity of protein GPR120 were observed, which was probably associated with the regulation of TAK1/IKK-α/IkB-α/p65 pathway. Our results indicated that triggering GPR120 via the inhibition of TAK1/IKK-α/IkB-α/p65 pathway might be an important target for Crohn's colitis.

Metformin Improves Ileal Epithelial Barrier Function in Interleukin-10 Deficient Mice.

Background and aimsThe impairment of intestinal epithelial barrier is the main etiologic factor of inflammatory bowel disease. The proper intestinal epithelial proliferation and differentiation is crucial for maintaining intestinal integrity. Metformin is a common anti-diabetic drug. The objective is to evaluate the protective effects of metformin on ileal epithelial barrier integrity using interleukin-10 deficient (IL10KO) mice.MethodsWild-type and IL10KO mice were fed with/without metformin for 6 weeks and then ileum was collected for analyses. The mediatory role of AMP-activated protein kinase (AMPK) was further examined by gain and loss of function study in vitro.ResultsCompared to wild-type mice, IL10KO mice had increased proliferation, reduced goblet cell and Paneth cell lineage differentiation in the ileum tissue, which was accompanied with increased crypt expansion. Metformin supplementation mitigated intestinal cell proliferation, restored villus/crypt ratio, increased goblet cell and Paneth cell differentiation and improved barrier function. In addition, metformin supplementation in IL10KO mice suppressed macrophage pro-inflammatory activity as indicated by reduced M1 macrophage abundance and decreased pro-inflammatory cytokine IL-1β, TNF-α and IFN-γ expressions. As a target of metformin, AMPK phosphorylation was enhanced in mice treated with metformin, regardless of mouse genotypes. In correlation, the mRNA level of differentiation regulator including bmp4, bmpr2 and math1 were also increased in IL10KO mice supplemented with metformin, which likely explains the enhanced epithelial differentiation in IL10KO mice with metformin. Consistently, in Caco-2 cells, metformin promoted claudin-3 and E-cadherin assembly and mitigated TNF-α-induced fragmentation of tight junction proteins. Gain and loss of function assay also demonstrated AMPK was correlated with epithelial differentiation and proliferation.ConclusionsMetformin supplementation promotes secretory cell lineage differentiation, suppresses inflammation and improves epithelial barrier function in IL10KO mice likely through activation of AMPK, showing its beneficial effects on gut epithelial.

Induction of Alternative Proinflammatory Cytokines Accounts for Sustained Psoriasiform Skin Inflammation in IL-17C+IL-6KO Mice

IL-6 inhibition has been unsuccessful in treating psoriasis, despite high levels of tissue and serum IL-6 in patients. In addition, de novo psoriasis onset has been reported after IL-6 blockade in patients with rheumatoid arthritis. To explore mechanisms underlying these clinical observations, we backcrossed an established psoriasiform mouse model (IL-17C+ mice) with IL-6-deficient mice (IL-17C+KO) and examined the cutaneous phenotype. IL-17C+KO mice initially exhibited decreased skin inflammation; however, this decrease was transient and reversed rapidly, concomitant with increases in skin Tnf, Il36α/β/γ, Il24, Epgn, and S100a8/a9 to levels higher than those found in IL-17C+ mice. A comparison of IL-17C+ and IL-17C+KO mouse skin transcriptomes with that of human psoriasis skin revealed significant correlation among transcripts of skin of patients with psoriasis and IL-17C+KO mouse skin, and confirmed an exacerbation of the inflammatory signature in IL-17C+KO mice that aligns closely with human psoriasis. Transcriptional analyses of IL-17C+ and IL-17C+KO primary keratinocytes confirmed increased expression of proinflammatory molecules, suggesting that in the absence of IL-6, keratinocytes increase production of numerous additional proinflammatory cytokines. These preclinical findings may provide insight into why patients with arthritis being treated with IL-6 inhibitors develop new onset psoriasis and why IL-6 blockade for the treatment of psoriasis has not been clinically effective.