Inflammation In Multiple Tissues Research Articles

AP-1 is a regulatory transcription factor of inflammaging in the murine kidney and liver.

Aging is characterized by chronic low-grade inflammation in multiple tissues, also termed "inflammaging", which represents a significant risk factor for many aging-related chronic diseases. However, the mechanisms and regulatory networks underlying inflammaging across different tissues have not yet been fully elucidated. Here, we profiled the transcriptomes and epigenomes of the kidney and liver from young and aged mice and found that activation of the inflammatory response is a conserved signature in both tissues. Moreover, we revealed links between transcriptome changes and chromatin dynamics through integrative analysis and identified AP-1 and ETS family transcription factors (TFs) as potential regulators of inflammaging. Further in situ validation showed that c-JUN (a member of the AP-1 family) was mainly activated in aged renal and hepatic cells, while increased SPI1 (a member of the ETS family) was mostly induced by elevated infiltration of macrophages, indicating that these TFs have different mechanisms in inflammaging. Functional data demonstrated that genetic knockdown of Fos, a major member of the AP-1 family, significantly attenuated the inflammatory response in aged kidneys and livers. Taken together, our results revealed conserved signatures and regulatory TFs of inflammaging in the kidney and liver, providing novel targets for the development of anti-aging interventions.

A Lipid Perspective on Regulated Pyroptosis.

Pyroptosis is a novel pro-inflammatory cell programmed death dependent on Gasdermin (GSMD) family-mediated membrane pore formation and subsequent cell lysis, accompanied by the release of inflammatory factors and expanding inflammation in multiple tissues. All of these processes have impacts on a variety of metabolic disorders. Dysregulation of lipid metabolism is one of the most prominent metabolic alterations in many diseases, including the liver, cardiovascular system, and autoimmune diseases. Lipid metabolism produces many bioactive lipid molecules, which are important triggers and endogenous regulators of pyroptosis. Bioactive lipid molecules promote pyroptosis through intrinsic pathways involving reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, lysosomal disruption, and the expression of related molecules. Pyroptosis can also be regulated during the processes of lipid metabolism, including lipid uptake and transport, de novo synthesis, lipid storage, and lipid peroxidation. Taken together, understanding the correlation between lipid molecules such as cholesterol and fatty acids and pyroptosis during metabolic processes can help to gain insight into the pathogenesis of many diseases and develop effective strategies from the perspective of pyroptosis.

OP11 Expanded genome-wide association study of Inflammatory Bowel Disease identifies 174 novel loci and directly implicates new genes in disease susceptibility

Abstract Background Genome-wide association studies (GWASs) have identified 243 loci associated with inflammatory bowel disease (IBD). However, the mapping of additional disease loci and causal variants is still limited by sample size. Larger GWAS can provide further insights into causal biology. Methods We performed a GWAS meta-analysis of 33 cohorts, totalling 54,439 IBD patients (N=30,574 with Crohn’s disease (CD), 21,193 with Ulcerative Colitis (UC)) and 37,054 European controls. Genotype imputation was undertaken using the TOPMed diverse population panel and association tests were performed using REGENIE. These results were meta-analysed with summary statistics from 4 additional studies, a Danish cohort, the UK Biobank, deCODE, and FinnGen, totalling 73,030 IBD patients and 1 million controls, Fig1. Results We identified 174 novel genome-wide significant signals (32 associated with CD, 36 with UC, 106 with IBD, Fig2). Of these, 79 are located >1Mb from any known GWAS loci. We also identified two new population specific genetic associations, Fig3. Six new loci contain genes implicated in monogenic syndromes that include colitis: CARMIL2, DOCK8, G6PC3, HPS4, NCF1, PIK3CD. Several new loci alter the expression of nearby genes, suggesting that aberrant expression of these genes underpins the association. For instance, a new variant associated with decreased risk of IBD increases the expression of VSIR in colon Fig4. VSIR knockout mice have increased expression of IL23 and develop chronic inflammation in multiple tissues. Fine-mapping analyses identified likely causal missense variants at three new loci. DOK2 (ORCD=1.3, 27p=2x10-12) encodes a protein expressed in macrophages and T cells. Loss of Dok2 in mice causes severe DSS-induced colitis with reduced IL17A and IL22 expression. The same variant has been recently associated with CD in a sequencing study. SHARPIN (ORCD=1.2, p=1x10-16) is part of the linear ubiquitin chain assembly complex that modulates activation of the NF-κB pathway. Loss-of-function (LOF) mutations in other proteins in the complex are associated with immunodeficiency and systemic autoinflammation. CARMIL2 (ORUC=1.2, p=1x10-10) is required for NF-κB signalling in both B and T cells. LOF mutations are associated with primary immunodeficiencies and paediatric forms of IBD. Conclusion The greatly expanded sample size in our latest GWAS meta-analysis has enabled the identification of low frequency variants with larger effects on IBD susceptibility than the more common variants typically identified by previous GWAS. The biological overlap between Mendelian and complex forms of IBD is demonstrated by the discovery of common non-coding variants associated with complex forms of IBD that dysregulate the function of a Mendelian IBD gene.

Bone Remodeling and the Role of TRAF3 in Osteoclastic Bone Resorption.

Skeletal health is maintained by bone remodeling, a process in which microscopic sites of effete or damaged bone are degraded on bone surfaces by osteoclasts and subsequently replaced by new bone, which is laid down by osteoblasts. This normal process can be disturbed in a variety of pathologic processes, including localized or generalized inflammation, metabolic and endocrine disorders, primary and metastatic cancers, and during aging as a result of low-grade chronic inflammation. Osteoclast formation and activity are promoted by factors, including cytokines, hormones, growth factors, and free radicals, and require expression of macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) by accessory cells in the bone marrow, including osteoblastic and immune cells. Expression of TNF receptor-associated factor 6 (TRAF6) is required in osteoclast precursors to mediate RANKL-induced activation of NF-κB, which is also necessary for osteoclast formation and activity. TRAF3, in contrast is not required for osteoclast formation, but it limits RANKL-induced osteoclast formation by promoting proteasomal degradation of NF-κB-inducing kinase in a complex with TRAF2 and cellular inhibitor of apoptosis proteins (cIAP). TRAF3 also limits osteoclast formation induced by TNF, which mediates inflammation and joint destruction in inflammatory diseases, including rheumatoid arthritis. Chloroquine and hydroxychloroquine, anti-inflammatory drugs used to treat rheumatoid arthritis, prevent TRAF3 degradation in osteoclast precursors and inhibit osteoclast formation in vitro. Chloroquine also inhibits bone destruction induced by ovariectomy and parathyroid hormone in mice in vivo. Mice genetically engineered to have TRAF3 deleted in osteoclast precursors and macrophages develop early onset osteoporosis, inflammation in multiple tissues, infections, and tumors, indicating that TRAF3 suppresses inflammation and tumors in myeloid cells. Mice with TRAF3 conditionally deleted in mesenchymal cells also develop early onset osteoporosis due to a combination of increased osteoclast formation and reduced osteoblast formation. TRAF3 protein levels decrease in bone and bone marrow during aging in mice and humans. Development of drugs to prevent TRAF3 degradation in immune and bone cells could be a novel therapeutic approach to prevent or reduce bone loss and the incidence of several common diseases associated with aging.

Role of Regulator of G‐Protein Signaling 5 Protein in Modulating Emotional Behaviors in the Absence and Presence of Angiotensin II‐Induced Hypertension

Angiotensin II (Ang II), through its actions on the Ang II type 1 receptor, a G‐protein coupled receptor (GPCR), increases blood pressure and causes oxidative stress and inflammation in multiple tissues, including in the cerebral vasculature. Ang II‐induced brain inflammation may also contribute to emotional abnormalities, such as anxiety‐ and depression‐like behavior. Regulator of G‐protein signaling (RGS) proteins modulate responses to extracellular signals (such as Ang II) acting through GPCRs by accelerating GTPase activity on Gα subunits, thus terminating G‐protein activity. RGS5, which is expressed in the brain and cerebral vasculature, mediates Ang II‐induced increases in blood pressure and vascular structure. In this study, we examined the role of RGS5 in modulating cerebral vascular oxidative stress, and anxiety‐ and depression‐like behavior in the absence and presence of elevated Ang II levels in adult (20–24 week old) male mice. Wild‐type (RGS5+/+) and RGS5‐deficient (RGS5−/−) mice were treated with vehicle (veh, saline) or Ang II (1 mg/kg/d) via osmotic minipumps for 21 days. Systolic blood pressure (SBP, measured using tail cuff), anxiety (measured using elevated plus maze) and depression (measured using tail suspension test)‐related behaviors were assessed in conscious mice; superoxide levels (measured using LO12 chemiluminesence) were examined in isolated cerebral vessels. Ang II treatment caused a greater increase in SBP in RGS5−/− compared to RGS5+/+ mice (ΔSBP at Day 17 in Ang II–treated RGS5+/+= 16±7mmHg, n=10; Ang II‐treated RGS5−/−= 37±5 mmHg, n=10, P<0.05), confirming that RGS5 modulates Ang II‐induced increases in blood pressure. In RGS5+/+ mice, Ang II treatment increased anxiety‐like behavior (time spent in open arms [secs] in veh‐treated RGS5+/+=79±12, n=7; Ang II‐treated=49±5, n=8, P<0.05). In vehicle‐treated RGS5−/− mice, anxiety‐like behavior was increased compared to vehicle‐treated RGS5+/+ mice (time spent in open arms [secs] in veh‐treated RGS5−/−=36±5, n=8, P<0.05 vs veh‐treated RGS5+/+), suggesting that RGS5 deficiency causes anxiety‐like behavior. There was no further effect of Ang II treatment on anxiety‐like behavior in RGS5−/− mice (time spent in open arms [secs] in Ang II‐treated RGS5−/−=44±7, n=9, P>0.05 vs veh‐treated RGS−/−). Ang II treatment, and RGS5 deficiency alone had no effect on depression‐like behavior (time immobilized [secs] in veh‐treated RGS5+/+=164±10, n=11; Ang II‐treated RGS5+/+=170±10, n=10, P>0.05 vs vehicle; veh‐treated RGS5−/−=178±12, n=10, P>0.05 vs veh‐treated RGS5+/+); however combined RGS5 deficiency and Ang II treatment resulted in an increase in depression‐like behavior (time immobilized [secs] in Ang II‐treated RGS5−/−=202±10, n=11, P<0.05 vs Ang II‐treated RGS5+/+), suggesting that combined RGS5 deficiency and Ang II treatment increased depression‐like behavior compared to Ang II treatment alone. There was no effect of Ang II treatment or RGS5 deficiency on cerebral vascular superoxide levels. Overall, these data suggest important roles for RGS5 in modulating Ang II‐induced increases in blood pressure, and anxiety‐ and depression‐like behavior.Support or Funding InformationRaabe College of Pharmacy, Ohio Northern UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polyphenol- and Caffeine-Rich Postfermented Pu-erh Tea Improves Diet-Induced Metabolic Syndrome by Remodeling Intestinal Homeostasis in Mice.

Postfermented Pu-erh tea (PE) protects against metabolic syndrome (MS), but little is known regarding its underlying mechanisms. Animal experiments were performed to determine whether the gut microbiota mediated the improvement in diet-induced MS by PE and its main active components (PEAC). We confirmed that PE altered the body composition and energy efficiency, attenuated metabolic endotoxemia and systemic and multiple-tissue inflammation, and improved the glucose and lipid metabolism disorder in high-fat diet (HFD)-fed mice via multiple pathways. Notably, PE promoted the lipid oxidation and browning of white adipose tissue (WAT) in HFD-fed mice. Polyphenols and caffeine (CAF) played critical roles in improving these parameters. Meanwhile, PE remodeled the disrupted intestinal homeostasis that was induced by the HFD. Many metabolic changes observed in the mice were significantly correlated with alterations in specific gut bacteria. Akkermansia muciniphila and Faecalibacterium prausnitzii were speculated to be the key gut bacterial links between the PEAC treatment and MS at the genus and species levels. Interestingly, A. muciniphila administration altered body composition and energy efficiency, promoted the browning of WAT, and improved the lipid and glucose metabolism disorder in the HFD-fed mice, whereas F. prausnitzii administration reduced the HFD-induced liver and intestinal inflammatory responses. In summary, polyphenol- and CAF-rich PE improved diet-induced MS, and this effect was associated with a remodeling of the gut microbiota.

αMSH inhibits adipose inflammation via reducing FoxOs transcription and blocking Akt/JNK pathway in mice.

Alpha melanocyte stimulating hormone (αMSH) abates inflammation in multiple tissues, while Forkhead box proteins O (FoxOs) stimulate inflammatory cascade. However, the relationship between αMSH and FoxOs in adipose inflammation remains unclear. In this study, we used LPS-induced inflammation model, attempted to interpret the function of αMSH in inflammation and the interactions with FoxOs. Results indicated that upon inflammatory situation, the secretion of αMSH and the expression of its receptor MC5R were greatly decreased, but FoxOs expressions were elevated. After the treatment with αMSH, LPS-induced adipose inflammation together with FoxOs expressions was significantly reduced. Conversely, when Foxo1, Foxo3a or Foxo4 overexpressed in αMSH treated inflammatory mouse model, all the anti-inflammatory impacts of αMSH were found disappeared. We further studied the mechanisms by which αMSH exerts its anti-inflammatory impacts and how FoxOs reverse αMSH's function. Foxo4 was found as a negative regulator for MC5R transcription in αMSH inhibited inflammation. Moreover, a negative role was found of αMSH in regulating both Akt and JNK signal pathways by observing the enhanced the anti-inflammatory impacts of pathway-specific inhibitors with αMSH treatment. Our findings demonstrate αMSH plays a key role in the prevention of adipose inflammation and inflammatory diseases by down-regulating Akt/JNK signal pathway and negatively interacting with FoxOs, which brings up αMSH as a novel candidate factor in the adipose anti-inflammation process in obesity.

NF-κB-dependent airway inflammation triggers systemic insulin resistance.

Inflammatory lung diseases (e.g., pneumonia and acute respiratory distress syndrome) are associated with hyperglycemia, even in patients without a prior diagnosis of Type 2 diabetes. It is unknown whether the lung inflammation itself or the accompanying comorbidities contribute to the increased risk of hyperglycemia and insulin resistance. To investigate whether inflammatory signaling by airway epithelial cells can induce systemic insulin resistance, we used a line of doxycycline-inducible transgenic mice that express a constitutive activator of the NF-κB in airway epithelial cells. Airway inflammation with accompanying neutrophilic infiltration was induced with doxycycline over 5 days. Then, hyperinsulinemic-euglycemic clamps were performed in chronically catheterized, conscious mice to assess insulin action. Lung inflammation decreased the whole body glucose requirements and was associated with secondary activation of inflammation in multiple tissues. Metabolic changes occurred in the absence of hypoxemia. Lung inflammation markedly attenuated insulin-induced suppression of hepatic glucose production and moderately impaired insulin action in peripheral tissues. The hepatic Akt signaling pathway was intact, while hepatic markers of inflammation and plasma lactate were increased. As insulin signaling was intact, the inability of insulin to suppress glucose production in the liver could have been driven by the increase in lactate, which is a substrate for gluconeogenesis, or due to an inflammation-driven signal that is independent of Akt. Thus, localized airway inflammation that is observed during inflammatory lung diseases can contribute to systemic inflammation and insulin resistance.

Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

Hematopoietic Tissue Factor–Protease-Activated Receptor 2 Signaling Promotes Hepatic Inflammation and Contributes to Pathways of Gluconeogenesis and Steatosis in Obese Mice

Failure to inhibit hepatic gluconeogenesis is a major mechanism contributing to fasting hyperglycemia in type 2 diabetes and, along with steatosis, is the hallmark of hepatic insulin resistance. Obesity is associated with chronic inflammation in multiple tissues, and hepatic inflammation is mechanistically linked to both steatosis and hepatic insulin resistance. Here, we delineate a role for coagulation signaling via tissue factor (TF) and proteinase-activated receptor 2 (PAR2) in obesity-mediated hepatic inflammation, steatosis, and gluconeogenesis. In diet-induced obese mice, TF tail signaling independent of PAR2 drives CD11b(+)CD11c(+) hepatic macrophage recruitment, and TF-PAR2 signaling contributes to the accumulation of hepatic CD8(+) T cells. Transcripts of key pathways of gluconeogenesis, lipogenesis, and inflammatory cytokines were reduced in high-fat diet-fed mice that lack the cytoplasmic domain of TF (F3) (TF(ΔCT)) or that are deficient in PAR2 (F2rl1), as well as by pharmacological inhibition of TF-PAR2 signaling in diet-induced obese mice. These gluconeogenic, lipogenic, and inflammatory pathway transcripts were similarly reduced in response to genetic ablation or pharmacological inhibition of TF-PAR2 signaling in hematopoietic cells and were mechanistically associated with activation of AMP-activated protein kinase (AMPK). These findings indicate that hematopoietic TF-PAR2 signaling plays a pivotal role in the hepatic inflammatory responses, steatosis, and hepatic insulin resistance that lead to systemic insulin resistance and type 2 diabetes in obesity.

Verapamil modulates interleukin‐5 and interleukin‐6 secretion in organotypic human sinonasal polyp explants

Verapamil is an L-type calcium channel blocker (CCB) that has been shown to have immunomodulatory properties in a variety of tissues. The goal of this study was determine whether verapamil is capable of regulating cytokine secretion in sinonasal polyps and to compare this effect to dexamethasone, an established immunosuppressive corticosteroid. This was an institutional review board (IRB)-approved study in sinonasal polyp explants derived from 8 patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Polyps were incubated with dexamethasone or verapamil for 24 hours followed by an additional 24 hours with Staphylococcal enterotoxin B (SEB). Concentrations of secreted cytokines over each exposure period were determined by enzyme-linked immunosorbent assay (ELISA) and are expressed as a percent. Results were compared using a 2-tailed Student t test. The percent of SEB-stimulated interleukin-5 (IL-5) secretion (mean ± standard deviation [SD], 339.94% ± 315.48%) between the second and first treatment periods was significantly reduced following exposure to dexamethasone (74.08% ± 26.77%, p < 0.05) and verapamil (119.99% ± 69.32%, p < 0.05). The percent of SEB-stimulated IL-6 secretion (217.53% ± 89.51%) was also significantly reduced following exposure to verapamil (148.82% ± 79.15%, p < 0.05) but not dexamethasone (148.86% ± 145.24%). Finally, the percent of SEB-stimulated thymic stromal lymphopoietin (TSLP) secretion (37.86% ± 18.88%) demonstrated a nonsignificant trend toward reduction with both dexamethasone (31.15% ± 35.28%) and verapamil (20.14% ± 12.10%). Although the mechanism has yet to be fully understood, L-type CCBs are capable of reducing inflammation in multiple tissues. Verapamil was specifically found to reduce airway goblet cell hyperplasia and eosinophilic infiltration in a murine asthma model. Our data support these findings suggesting that verapamil can modulate T-helper cell type 2 (Th2)-associated cytokine secretion in sinonasal polyp explants. This data points to a possible therapeutic role for CCBs in the management of CRSwNP.

MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.

Progesterone suppresses the mTOR pathway and promotes generation of induced regulatory T cells with increased stability

While induced FoxP3(+) T cells (iTreg cells) are promising cellular therapeutics to treat inflammatory diseases, a limitation in utilizing iTreg cells prepared in vitro is their low stability in inflammatory conditions. Progesterone (P4) is an immune regulatory nuclear hormone with a potent Treg induction activity. We reasoned that this function of progesterone would be utilized to generate iTreg cells with highly suppressive activity and improved stability in vivo. Here we generated iTreg cells with progesterone in vitro and found that progesterone generates iTreg cells that are highly stable in inflammatory conditions. Moreover, P4-induced iTreg cells highly express latency-associated peptide TGF-β1 and are efficient in regulating inflammation in multiple tissues, whereas control iTreg cells induced with TGF-β1 alone are less stable and ineffective in suppressing inflammation. The function of progesterone in inducing iTreg cells with improved regulatory activity is associated with the function of P4 in suppressing the mTOR pathway. Moreover, the function of progesterone in inducing FoxP3(+) T cells is decreased but not completely abolished on nuclear progesterone receptor-deficient T cells, suggesting that both nuclear and nonnuclear progesterone receptors are involved in mediating the function. We conclude that P4 can be utilized to generate iTreg cells with a high therapeutic potential in treatment of tissue inflammation.

CCR7 Deficiency in NOD Mice Leads to Thyroiditis and Primary Hypothyroidism

CCR7 is involved in the initiation of immune responses and has been recently implicated in the control of tolerance. To analyze the role of CCR7 in autoimmunity, we backcrossed CCR7(ko/ko) mice (in which ko signifies deficient) onto the autoimmune-prone NOD background. Surprisingly, NODCCR7(ko/ko) mice never developed diabetes, but showed severe inflammation in multiple tissues including thyroid, lung, stomach, intestine, uterus, and testis. NODCCR7(ko/ko) mice had a marked enlargement of the thyroid gland (goiter) that was associated with circulating autoantibodies against thyroglobulin, and development of primary hypothyroidism (decreased levels of serum thyroxin, and augmented levels of thyroid-stimulating hormone in the pituitary gland), features found in Hashimoto's thyroiditis. Cells isolated from diseased thyroids and activated splenocytes from NODCCR7(ko/ko) animals induced goiter in NOD.SCID recipients, demonstrating that autoreactive cells were generated in the absence of CCR7. Moreover, thyroid disease could be accelerated in young NODCCR7(ko/ko) mice by immunization with thyroglobulin. These results demonstrate the complexity in the generation of multiple autoimmune phenotypes in NOD mice and indicate that CCR7 is a key molecule in their development.

Coactivator function of RIP140 for NFκB/RelA-dependent cytokine gene expression

AbstractInflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor κB (NFκB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks, but we have found that genetic as well as acute deficiency of RIP140 leads to the inhibition of the proinflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFκB subunit RelA and histone acetylase cAMP-responsive element binding protein (CREB)-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.

PD-1 deficiency reveals various tissue-specific autoimmunity by H-2band dose-dependent requirement of H-2g7for diabetes in NOD mice

Although many autoimmune diseases are associated with particular HLA/H-2 haplotypes, the mechanisms through which specific HLA/H-2 haplotypes afford autoimmune susceptibility remain enigmatic. Here, we analyzed the effects of the diabetes-associated (H-2(g7)) and an antidiabetogenic (H-2(b)) H-2 haplotypes in NOD mice deficient for programmed cell death-1 (PD-1, Pdcd1), a unique model of type 1 diabetes that confers complete penetrance and rapid onset of the disease. NOD-H2(b/b)Pdcd1(-/-) mice were completely protected from diabetes, confirming that H-2(g7) is indispensable for diabetes even in the absence of PD-1. However, NOD-H2(b/b)Pdcd1(-/-) mice developed autoimmune inflammation in multiple tissues including peripheral nerves, stomachs, and exocrine tissues, demonstrating that autoreactive T cells are generated in the context of H-2(b). These autoreactive T cells damaged target tissues only in the absence of PD-1, confirming that PD-1 deficiency unravels the hidden autoimmune susceptibility of the strain by reducing the threshold of T cell activation. Transfer experiments revealed that CD4 T cells are the effector cells of neuritis, and nerve-infiltrating CD4 T cells are strongly deviated toward Th1. Interestingly, neuritogenic T cells were also generated in the context of H-2(g7), in sharp contrast to the strict requirement of H-2(g7) for diabetes. In addition, 60% of the NOD-H2(b/g7)Pdcd1(-/-) mice developed diabetes, suggesting that H-2(b) does not dominantly suppress diabetes and that H-2(g7) induces diabetes in a dose-dependent fashion.

Pathology of Immunodeficient Mice With Naturally Occurring Murine Norovirus Infection

Murine norovirus (MNV) was recently discovered in Rag2-/-/Stat1-/- mice in a U.S. medical research facility. Presently, little is known concerning the epidemiology and natural history of this virus. We studied the pathology of naturally occurring MNV infection in 28 immunodeficient mice of several different genotypes (Rag1-/-/IFNgamma R-/-, OT1 Rag1-/-/IFNgamma R-/-, OT2 Rag1-/-/IFNgamma R-/-, Rag1-/-/Stat1-/-, and Rag2-/-) that were maintained in two U.S. research facilities. The mice were selected for study because sentinel mice housed in their holding rooms had been identified as positive for MNV-specific antibodies during routine screening for infectious agents. Our data indicate that in certain lines of immunodeficient mice, MNV can establish a disseminated infection that is characteristically associated with inflammation in multiple tissues, including liver (hepatitis), lung (focal interstitial pneumonia) and the peritoneal and pleural cavities. In addition, MNV can establish an asymptomatic infection in the mesenteric lymph nodes of Rag2-/- mice. Further studies are needed to determine whether MNV presents a confounding variable in immunological, toxicological and pathological studies in mice naturally infected with MNV.

Characterization of the Inflammatory Response to a Highly Selective PDE4 Inhibitor in the Rat and the Identification of Biomarkers that Correlate with Toxicity

The primary toxicity associated with repeated oral administration of the PDE4 inhibitor IC542 to the rat is an inflammatory response leading to tissue damage primarily in the gastrointestinal tract and mesentery. Although necrotizing vasculitis is frequently seen with other PDE4 inhibitors, blood vessel injury was rare following IC542 administration and was always associated with inflammation in the surrounding tissue. The incidence and severity of the histologic changes in these studies correlated with elevated peripheral blood leukocytes, serum IL-6, haptoglobin, and fibrinogen, and with decreased serum albumin. By monitoring haptoglobin, fibrinogen and serum albumin changes in IC542-treated rats, it was possible to identify rats with early histologic changes that were reversible. Since PDE4 inhibition is generally associated with anti-inflammatory activity, extensive inflammation in multiple tissues was unexpected with IC542. Co-administration of dexamethasone completely blocked IC542-induced clinical and histologic changes in the rat, confirming the toxicity resulted from inflammatory response. In addition, IC542 augmented release of the proinflammatory cytokine IL-6 in LPS-activated whole blood from rats but not monkeys or humans. The effect of IC542 on IL-6 release from rat leukocytes in vitro is consistent with the proinflammatory response observed in vivo and demonstrates species differences to PDE4 inhibition.

Increased expression of type 2 cytokines in chronic proliferative dermatitis (cpdm) mutant mice and resolution of inflammation following treatment with IL-12.

Chronic proliferative dermatitis (cpdm) is a spontaneous mutation that results in eosinophilic inflammation in multiple tissues, including the skin. To determine the mechanisms underlying the eosinophilic inflammation, the expression of cytokines in the skin was determined. There was increased expression of IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor in the skin of cpdm/cpdm mice, and no change in IL-10 and TNF expression. Supernatants of cultured spleen cells of cpdm/cpdm mice contained an increased amount of IL-5 and IL-13, and a decreased amount of IFN-gamma. The ability of the cpdm/cpdm mice to mount a delayed-type hypersensitivity response was greatly reduced. These data are consistent with impaired type 1 and excessive type 2 cytokine production in cpdm/cpdm mice. The significance of this imbalanced cytokine production was evident in the efficacy of systemic treatment of cpdm/cpdm mice with IL-12. Mutant mice treated for 3 weeks with IL-12 had minimal changes in the skin as opposed to the severe dermatitis in mice treated with the vehicle. Treatment with IL-11, which opposes the effect of IL-12, had no effect.

Strain and sex variation in the susceptibility to streptococcal cell wall-induced polyarthritis in the rat.

Sixteen inbred rat strains were examined for susceptibility and resistance to group A streptococcal cell wall-induced polyarthritis. The findings indicated that 2 or more genetic loci, as well as sex-related factors, played a major role in determining susceptibility to arthritis in this model. Breeding studies demonstrated that susceptibility was a dominant or codominant trait. A positive association between the severity of arthritis and the development of chronic inflammation in multiple tissues was also observed. In strains that were relatively resistant to arthritis, chronic inflammation was generally limited to the spleen. Since translocation of the poorly degradable and phlogogenic streptococcal cell walls to the synovium and other tissues appears to initiate inflammation, these studies suggested that susceptibility might be the result of a defect in host mechanisms limiting cell wall dissemination.