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Purpose: Pancreatic ductal adenocarcinoma (PDA) is associated with an immunosuppressive microenvironment that supports the growth of the malignancy as well as immune system evasion. Here we examine markers of immunosuppression in PDA within the context of the glycolytic tumor microenvironment, their interrelationship with tumor biology and association with overall survival.Experimental Design: We utilized tissue microarrays consisting of 223 PDA patients annotated for clinical stage, tumor size, lymph node involvement, and survival. Expression of CD163, FoxP3, PD-L1, and MCT4 was assessed by IHC and statistical associations were evaluated by univariate and multivariate analysis. Multimarker subtypes were defined by random forest analysis. Mechanistic interactions were evaluated using PDA cell lines and models for myeloid differentiation.Results: PDA exhibits discrete expression of CD163, FoxP3, and PD-L1 with modest individual significance. However, combined low expression of these markers was associated with improved prognosis (P = 0.02). PDA tumor cells altered macrophage phenotype and function, which supported enhanced invasiveness in cell-based models. Lactate efflux mediated by MCT4 was associated with, and required for, the selective conversion of myeloid cells. Correspondingly, MCT4 expression correlated with immune markers in PDA cases, and increased the significance of prognostic subtypes (P = 0.002).Conclusions: There exists a complex interplay between PDA tumor cells and the host immune system wherein immunosuppression is associated with negative outcome. MCT4 expression, representative of the glycolytic state of PDA, contributes to the phenotypic conversion of myeloid cells. Thus, metabolic status of PDA tumors is an important determinant of the immunosuppressive environment. Clin Cancer Res; 22(14); 3606–17. ©2016 AACR.

Analysis of lymphocyte and neutrophil infiltration in the TMA cohort and the impact of these factors on patient survival.

Statistical analysis of data used to generate Kaplan-Meier curves. Table S1 Cox proportional hazards analysis of indicated FoxP3 expression Table S2 Multivariable Cox proportional hazards analysis of FoxP3 expression and other variables Table S3 Cox proportional hazards analysis of indicated CD163 expression Table S4 Multivariable Cox proportional hazards analysis of CD163 expression and other variables Table S5 Cox proportional hazards analysis and median survival of indicated markers Table S6 Cox proportional hazards analysis of indicated PDL1 expression Table S7 Multivariable Cox proportional hazards analysis of PDL1 expression and other variables Table S8 Cox proportional hazards analysis and median survival of indicated markers Table S9 Cox proportional hazards analysis and median survival of indicated markers Table S10 Cox proportional hazards analysis and median survival of indicated clusters Table S11 Cox proportional hazards analysis and median survival of indicated clusters Table S12 Cox proportional hazards analysis and median survival of indicated clusters Table S13 Cox proportional hazards analysis and median survival of indicated clusters Table S14 Multivariable Cox proportional hazards analysis of clusters and other variables

Background: Autoimmune diseases, such as systemic lupus erythematosus, can have severe impacts on quality of life. They are one of the leading causes of death for women in the United States. Distinguished by the body damaging its own tissues and organs, they are often classified and diagnosed based on autoantibody levels. Treatments often include immunosuppressant drugs, which can have adverse effects. Aim of Study: Amaranth is a good functional food candidate, possessing antioxidants, bioactive compounds, and a variety of health benefits, such as lowering cholesterol, and aiding diabetes and hypertension. Previous studies have largely focused on the grain or seed, but amaranth oil is less explored. This study examines whether orally administered amaranth oil had any effects on autoantibodies and splenic immune cell populations in murine subjects. Methods: Mice in the experimental group (n = 3) were given 4μl of amaranth oil per gram of mouse weight for 5 days a week over 84 days. Control mice (n = 2) were sham treated on the same schedule with no oil. To determine autoantibody levels, enzyme-linked immunosorbent assays (ELISAs) were first conducted on wells pre-coated with double stranded DNA, single stranded DNA, histones, or double stranded DNA and then histones (nucleosomes). Autoantibody presence was quantified by measuring absorbance at 405nm. Splenic cell populations were examined with flow cytometry and compared using a student’s t-test. Results: Compared to the control group, the mice receiving amaranth oil showed decreased IgG and IgM histone autoantibody absorbance levels throughout the whole study. IgG dsDNA, ssDNA, and nucleosome autoantibody absorbances were lower than that of the control group for the first 42 days. IgM dsDNA, ssDNA, and nucleosome autoantibody absorbances were lower only for the first 14 days. There were no significant differences found amongst splenic immune cell populations between the control and experimental groups. Conclusion: These preliminary data show that amaranth oil may help decrease autoantibody levels in lupus prone murine subjects. However, given the small number of subjects in this study, further research is needed to confirm observed effects and determine the most effective dose and administration schedule. Keywords: autoantibody, IgG, IgM, histone, dsDNA, ssDNA, nucleosome, amaranth oil, immunoglobulin, lupus

Background : Kidney disease is a leading cause of death in the United States and the world. Proteinuria signifies kidney damage and can exacerbate kidney disease. It has become an important indicator of kidney disease; reducing proteinuria results in renoprotective effects and slows renal disease. Existing treatments do not work for every patient. Aim of Study : Amaranth is a tropical plant that was regularly consumed in the ancient Central American diet. It has a plethora of health effects and is a strong functional food candidate. This study examines whether a regular oral dose of amaranth oil decreased proteinuria concentration in murine subjects. Methods : Mice in the experimental group (n = 3) were given 4μl of amaranth oil per gram of mouse weight for 5 days a week over 84 days. Control mice (n = 2) were sham treated on the same schedule with no oil. Urine protein concentration was determined by Bradford assay, measuring absorbance at 595nm, then comparing with a BSA standard curve. Results : The experimental group showed decreased proteinuria levels throughout the entire 84 days of study Conclusion : Results show amaranth oil may help decrease proteinuria levels in lupus prone murine subjects. Given the small sample size, the data are preliminary. More research is needed to validate the results and determine the optimal dose and treatment schedule. Key words: proteinuria, amaranth oil, renoprotective, kidney disease, renal disease, lupus, functional food

Tsai et al. demonstrate that loss of Bim (BCL2L11) in myeloid cells in mice (LysMCreBimfl/fl) is sufficient to induce systemic autoimmunity. Kidney macrophages in LysMCreBimfl/fl mice possess a proinflammatory transcriptional signature and signal through TRIF to cause end-stage glomerulonephritis., The Bcl-2 family is considered the guardian of the mitochondrial apoptotic pathway. We demonstrate that Bim acts as a molecular rheostat by controlling macrophage function not only in lymphoid organs but also in end organs, thereby preventing the break in tolerance. Mice lacking Bim in myeloid cells (LysMCreBimfl/fl) develop a systemic lupus erythematosus (SLE)–like disease that mirrors aged Bim−/− mice, including loss of marginal zone macrophages, splenomegaly, lymphadenopathy, autoantibodies (including anti-DNA IgG), and a type I interferon signature. LysMCreBimfl/fl mice exhibit increased mortality attributed to glomerulonephritis (GN). Moreover, the toll-like receptor signaling adaptor protein TRIF (TIR-domain–containing adapter-inducing interferon-β) is essential for GN, but not systemic autoimmunity in LysMCreBimfl/fl mice. Bim-deleted kidney macrophages exhibit a novel transcriptional lupus signature that is conserved within the gene expression profiles from whole kidney biopsies of patients with SLE. Collectively, these data suggest that the Bim may be a novel therapeutic target in the treatment of SLE.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is associated with an immunosuppressive microenvironment that supports the growth of the malignancy as well as immune system evasion. Here we examine markers of immunosuppression in PDA within the context of the glycolytic tumor microenvironment, their interrelationship with tumor biology and association with overall survival. Experimental Design: We utilized tissue microarrays consisting of 223 PDA patients annotated for clinical stage, tumor size, lymph node involvement, and survival. Expression of CD163, FoxP3, PD-L1, and MCT4 was assessed by IHC and statistical associations were evaluated by univariate and multivariate analysis. Multimarker subtypes were defined by random forest analysis. Mechanistic interactions were evaluated using PDA cell lines and models for myeloid differentiation. Results: PDA exhibits discrete expression of CD163, FoxP3, and PD-L1 with modest individual significance. However, combined low expression of these markers was associated with improved prognosis (P = 0.02). PDA tumor cells altered macrophage phenotype and function, which supported enhanced invasiveness in cell-based models. Lactate efflux mediated by MCT4 was associated with, and required for, the selective conversion of myeloid cells. Correspondingly, MCT4 expression correlated with immune markers in PDA cases, and increased the significance of prognostic subtypes (P = 0.002). Conclusions: There exists a complex interplay between PDA tumor cells and the host immune system wherein immunosuppression is associated with negative outcome. MCT4 expression, representative of the glycolytic state of PDA, contributes to the phenotypic conversion of myeloid cells. Thus, metabolic status of PDA tumors is an important determinant of the immunosuppressive environment. Clin Cancer Res; 22(14); 3606–17. ©2016 AACR.

Curcumin is known to have immunomodulatory potential in addition to anti-oxidant, anti-inflammatory and anti-carcinogenic effects. The aim of the present study is to investigate the therapeutic effects of curcumin on immune-mediated renal disease in an anti-glomerular basement membrane (GBM) model (representing acute kidney Injury, AKI) and murine lupus model (representing chronic kidney disease, CKD). In the AKI model, female anti-GBM 129/svj mice were administered with curcumin right before disease induction. In the CKD model, female MRL.lpr mice at the age of 8-10 weeks old were treated with curcumin or placebo via oral gavage daily for two months. After treatment, serum autoantibody levels, splenomegaly and spleen cellularity were reduced in murine lupus. Collectively, curcumin ameliorated kidney disease in the two mouse models with either acute or chronic nephritis, as marked by reduced proteinuria, blood urea nitrogen, glomerulonephritis, crescent formation, tubule-interstitial disease, and renal infiltration by lymphocytes. In addition, curcumin treatment reduced activation of the NFkB, MAPK, AKT and pBAD pathways either systemically, or within the inflamed kidneys. These findings suggest that natural food supplements could become an alternative approach to ameliorating immune-mediated kidney diseases.

Over the past few decades, our understanding of the role of adipose tissue has changed dramatically. Far from simply being a site of energy storage or a modulator of the endocrine system, adipose tissue has emerged as an important regulator of multiple important processes including inflammation. Adipokines are a diverse family of soluble mediators with a range of specific actions on the immune response. Autoimmune diseases are perpetuated by chronic inflammatory responses but the exact etiology of these diseases remains elusive. While researchers continue to investigate these causes, millions of people continue to suffer from chronic diseases. To this end, an increased interest has developed in the connection between adipose tissue-secreted proteins that influence inflammation and the onset and perpetuation of autoimmunity. This review will focus on recent advances in adipokine research with specific attention on a subset of adipokines that have been associated with autoimmune diseases.

Recently developed potent and selective CDK4/6 inhibitors fall into two classes based on structure and toxicity profiles in clinical studies. One class, exemplified by palbociclib and ribociclib, exhibits neutropenia as a dose-limiting toxicity and requires discontinuous dosing. In contrast, the structurally distinct CDK4/6 inhibitor abemaciclib is dosed continuously, and has diarrhea and fatigue as dose-limiting toxicities. In preclinical models, palbociclib has been extensively studied and induces cell cycle inhibition in an RB-dependent manner. Thus far, abemaciclib has been less extensively evaluated. We found that abemaciclib cell cycle inhibitory activity is RB-dependent at clinically achievable concentrations. Abemaciclib elicited potent suppression of RB/E2F regulated genes associated with prognosis in ER-positive breast cancer. However, unlike palbociclib, at 250nM-1 µM doses abemaciclib induced cell death in RB-deficient cell lines. This response was associated with a rapidly-induced multi-vacuolar phenotype indicative of lysosomal membrane permeabilization that could be ameliorated with chloroquine. This event was not a reflection of inhibition of other CDK family members, but could be recapitulated with CBX4945 that inhibits casein and DYRK/HIPK kinases. To determine if these "off-target" features of abemaciclib were observed in vivo, mice harboring matched RB-positive and negative xenografts were treated with palbociclib and abemaciclib. In vivo, all of the apparent activity of abemaciclib was RB-dependent and strongly elicited suppression of cell cycle regulatory genes in a fashion markedly similar to palbociclib. Using gene expression data from cell lines and tumors treated with abemaciclib and palbociclib a composite signature of response to CDK4/6 inhibition was developed that included many genes that are individually required for tumor cell proliferation or viability. These data indicate that while abemaciclib and palbociclib can exert distinct biological and molecular effects, there are common gene expression features that could be broadly utilized in measuring the response to CDK4/6 inhibition.

Cancers mediated by viral etiology must exhibit deregulated cellular proliferation and evade immune recognition. The role of the retinoblastoma tumor suppressor (RB) pathway, which is lost at relatively high frequency in hepatocellular carcinoma (HCC), has recently been expanded to include the regulation of innate immune responsiveness. In this study we investigated the coordinate impact of RB-loss on cell cycle control and immune function in the liver. We found that RB depletion in hepatoma cells resulted in a compromised immunological response to multiple stimuli and reduced the potential of these cells to recruit myeloid cells. Viral-mediated liver-specific RB deletion in vivo led to the induction of genes associated with proliferation and cell cycle entry as well as the significant attenuation of genes associated with immune function, as evidenced by decreases in cytokine and chemokine expression, leukocyte recruitment, and hepatic inflammation. To determine if these changes in gene expression were instructive in human disease, we compared our liver-specific RB-loss gene signature to existing profiles of HCC and found that this signature was associated with disease progression and confers a worse prognosis. Conclusion: Our data confirm that RB participates in the regulation of innate immunity in liver parenchymal cells both in vitro and in vivo and to our knowledge describes the first gene signature associated with HCC that includes both immunoregulatory and proliferative genes and that can also be attributed to the alteration of a single gene in vitro. (Hepatology 2014;60:1231–1240)

Diverse etiologic events are associated with the development of hepatocellular carcinoma. During hepatocarcinogenesis, genetic events likely occur that subsequently cooperate with long-term exposures to further drive the progression of hepatocellular carcinoma. In this study, the frequent loss of the retinoblastoma (RB) tumor suppressor in hepatocellular carcinoma was modeled in response to diverse hepatic stresses. Loss of RB did not significantly affect the response to a steatotic stress as driven by a methionine- and choline-deficient diet. In addition, RB status did not significantly influence the response to peroxisome proliferators that can drive hepatomegaly and tumor development in rodents. However, RB loss exhibited a highly significant effect on the response to the xenobiotic1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene. Loss of RB yielded a unique proliferative response to this agent, which was distinct from both regenerative stresses and genotoxic carcinogens. Long-term exposure to 1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene yielded profound tumor development in RB-deficient livers that was principally absent in RB-sufficient tissue. These data demonstrate the context specificity of RB and the key role RB plays in the suppression of hepatocellular carcinoma driven by xenobiotic stress.

Catheters are implanted into the peritoneal cavity during the process of peritoneal dialysis. Though these catheters may be effective and beneficial, the impact of catheters on the immune system is poorly understood. Catheters and other devices implanted in the peritoneal cavity elicit a foreign body reaction. However, the immunological consequences of this remain uncharacterized. To model this, catheters were implanted into the peritoneal cavity of healthy mice. Catheter implantation induced rapid cellular changes within the peritoneal cavity. Whereas B-cells and T-cells were reduced, catheter implantation was associated with the rapid expansion of F4/80-low-positive, CD11b-positive macrophages that elaborated IL-10, and suppressed T-cell division and Th1 skewing in co-culture assays. Peritoneal catheter elicited macrophages had increased Jmjd3 but reduced NF-κB activation, and their emergence was MyD88-dependent. Collectively, these studies indicate that foreign body implantation into the peritoneal cavity is associated with the expansion of suppressor macrophages. Whether peritoneal cavity catheter implantation may have systemic immunoregulatory roles remains to be explored.

Autoimmunity occurs through a break in tolerance, which is considered to be mediated by a failure to delete autoreactive immune cells. The central mechanism for deleting cells is controlled by the apoptotic machinery. Apoptosis occurs via two distinct pathways, an extrinsic pathway involving transduction of an apoptotic signal following aggregation of a death receptor, such as Fas, to its ligand, Fas ligand (FasL), and an intrinsic pathway that signals through the mitochondria and is regulated by the bcl-2 family. In the extrinsic pathway, binding of homotrimeric FasL to Fas facilitates recruitment of both Fas-associated death domain protein (FADD) and pro-caspase-8 leading to the activation of caspase-8 and subsequent degradative phase of apoptosis (1). This process may be inhibited by cellular FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein (cFLIP), which is also recruited by FADD and acts as an endogenous suppressor of the Fas pathway (1). Mice mutant for Fas (lpr) mice show normal postnatal development, but over time exhibit splenomegaly, lymphadenopathy, hypergammaglobulinemia, autoantibodies, glomerulonephritis, and the presence of double negative T-cells (CD3+CD4-CD8-B220+), with disease severity highly dependent upon strain background (2). The advent of conditional deletion of genes has yielded novel, unexpected, and controversial results regarding specific effects of loss of Fas within the various hematopoietic cell populations. While previous studies have extensively examined the role that Fas plays in lymphocytes and dendritic cells (3-5), few or no studies have directly examined the cell-intrinsic role of Fas in myeloid cells and its contribution to autoimmunity. Here, mice were generated that conditionally lack Fas only in the myeloid cell compartment (CreLysMFasflox/flox). These mice displayed a disruption in monocyte/macrophage and granulocyte homeostasis that led to altered activation of dendritic cells and lymphocytes in a paracrine fashion. Moreover, CreLysMFasflox/flox mice presented with splenomegaly, as well as autoantibodies and proinflammatory cytokines/chemokines. Furthermore, deposited immune complexes in kidneys and glomerulonephritis were observed in mice with myeloid cell-specific loss of Fas. Taken together, these data indicate that loss of Fas in myeloid cells is sufficient to induce an inflammatory phenotype in mice reminiscent of SLE-like disease, suggesting a role of specifically targeting Fas in its treatment.

Objective Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies which activate the apoptotic cascade may have potential as a future therapy for RA, however few therapeutics fit this category. Recently, therapies that mimic the action of Bcl-2 homology 3 (BH3) domain-only proteins such as Bim have shown success in preclinical studies of cancer but their potential in autoimmune disease is unknown.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

The process of thymopoiesis is tightly regulated by a series of selection events which ensure that only functional T-lymphocytes directed against foreign antigens are exported into the periphery. The adaptive immune response largely depends on the regulation of thymocyte development, and thymocytes which fail selection in the thymus are removed by apoptosis. However, the roles of specific apoptotic proteins in early T-lymphocyte development are poorly understood. Here, we report a novel function for Bim in thymocyte development. There is an accumulation of thymocytes in Bim −/− mice that lack expression of CD4, CD8, CD44, and CD25 but express CD3 and TCR β . Further, the CD4 − CD8 − CD25 − CD44 − CD3 + TCR β + thymocytes are smaller and do not proliferate. These data suggest that these thymocytes are mature DN thymocytes that may have down-regulated the expression of CD4 and CD8. The DN thymocyte phenotype in Bim −/− mice is unaffected by the additional loss of Bak or Bax and is similar to the thymic phenotype in mice lacking both Bak and Bax. These data demonstrate that Bim functions to ensure the proper homeostasis of mature thymocytes during selection and thymic export.

Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

Introduction Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. Methods CreLysMCasp8fl/fl mice were bred via a cross between Casp8fl/fl mice and CreLysM mice, and RIPK3−/−CreLysMCasp8fl/fl mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. CreLysMCasp8fl/fl mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann–Whitney U test. Results Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8–deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8–deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6ChighCD11b+F4/80+ splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in CreLysMCasp8fl/fl mice. Further, caspase-8–deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. Conclusions These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0794-z) contains supplementary material, which is available to authorized users.

Objective: Rheumatoid arthritis (RA) is characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. Since there are only a few detectable cells undergoing apoptosis in the joint, it is possible that a defect in apoptosis may contribute to synovial hyperplasia. This study sought to identify and characterize the direct role of apoptotic regulators in a mouse model of inflammatory arthritis. Methods Using a serum transfer model, experimental arthritis was induced in mice lacking the proapoptotic Bcl-2 family genes Bak (Bak−/−), Bax (Bax−/−), or Bim (Bim−/−), as compared with wild-type (WT) control mice. Physical examination for edema of the ankles and histopathologic analysis of ankle sections were used to determine the severity of arthritis. The serum and ankles were examined for production of chemokines and cytokines using enzyme-linked immunosorbent or Luminex-based assays. Results Bim−/− mice displayed increased severity and prolongation of arthritis. In contrast, Bak−/− and Bax−/− mice showed no difference in the severity of arthritis as compared with WT mice. In addition, Bim−/− mice had elevated levels of proinflammatory chemokines and cytokines, decreased joint and serum production of antiinflammatory cytokines, fewer TUNEL-positive cells, and reduced levels of active caspase 3 as compared with WT mice. Conclusion These studies are the first to demonstrate a role for the proapoptotic Bcl-2 protein Bim in the effector phase of RA. The findings indicate that Bim potentially functions to repress the effector phase of arthritis by regulating the milieu of the joint and serum, and by inducing apoptosis.

One of the central functions of cyclin-dependent kinase inhibitors, such as p21, p27, or p16, is to prevent entry into the cell cycle. However, the question remains as to whether they have other functions in the cell. We previously demonstrated that overexpression of p21 in fibroblasts isolated from patients with rheumatoid arthritis decreases the production of pro-inflammatory molecules. Overexpression of p21 has been also shown to reduce the development of experimental arthritis in mice and rats. To explore the role of endogenous p21 in the development of arthritis, we induced arthritis in p21(-/-) mice using the K/BxN serum transfer model of arthritis. Mice deficient in p21 were more resistant to serum transfer-induced arthritis (K/BxN) than wild-type (wt) control mice. Fewer macrophages were detected in p21(-/-) as compared to wt joints following transfer of K/BxN serum. Chemotaxis assays of bone marrow-derived macrophages from p21(-/-) and wt mice revealed no difference in migration. However, there was a substantial decrease in inflammatory monocytes circulating in peripheral blood and in monocyte precursors in bone marrow of p21(-/-) mice as compared to wt mice. Adoptive transfer of wt bone marrow-derived macrophages into p21(-/-) mice restored the sensitivity to serum transfer-induced arthritis. These data suggest a novel role for p21 in regulating the development and/or differentiation of monocytic populations that are crucial for the induction of inflammatory arthritis.

The proapoptotic members of the Bcl-2 family can be subdivided into members that contain several Bcl-2 homology (BH) domains and those that contain only the BH3 domain. Although it is known that BH3-only proteins and the multi-BH domain proteins, Bak and Bax, are essential for programmed cell death, the overlapping role of these two subgroups has not been examined in vivo. To investigate this, we generated Bak/Bim and Bax/Bim double deficient mice. We found that although Bax−/−Bim−/−, but not Bak−/−Bim−/−, mice display webbed hind and front paws and malocclusion of the incisors, both groups of mice present with dysregulated hematopoiesis. Combined loss of Bak and Bim or Bax and Bim causes defects in myeloid and B-lymphoid development that are more severe than those found in the single knock-out mice. Bak−/−Bim−/− mice have a complement of thymocytes that resembles those in control mice, whereas Bax−/−Bim−/− mice are more similar to Bim−/− mice. However, thymocytes isolated from Bak−/−Bim−/− or Bax−/−Bim−/− mice are markedly more resistant to apoptotic stimuli mediated by the intrinsic pathway as compared with thymocytes from single-knockout mice. These data suggest an essential overlapping role for Bak or Bax and Bim in the intrinsic apoptotic pathway.

The retinoblastoma tumor suppressor (RB) was the first identified tumor suppressor based on germline predisposition to the pediatric eye tumor. Since these early studies, it has become apparent that the functional inactivation of RB is a common event in nearly all human malignancy. A great deal of research has gone into understanding how the loss of RB promotes tumor etiology and progression. Since malignant tumors are characterized by aberrant cell division, much of this research has focused upon the ability of RB to regulate the cell cycle by repression of proliferation-related genes. However, it is progressively understood that RB is an important mediator of multiple functions. One area that is gaining progressive interest is the emerging role for RB in regulating diverse features of immune function. These findings suggest that RB is more than simply a regulator of cellular proliferation; it is at the crossroads of proliferation and the immune response. Here we review the data related to the functional roles of RB on the immune system, relevance to immune evasion, and potential significance to the response to immune-therapy.

Summary Systemic lupus erythematosus (SLE) and lupus nephritis (LN) have strong concomitance with cardiovascular disease that cannot be explained fully by typical risk factors. We examined the possibility that serum or urine expression of adipokines may act as biomarkers for LN, as these proteins have been associated previously with cardiovascular disease as well as SLE. Antibody arrays were performed on serum and urine from lupus patients and matched controls using a cross-sectional study design. From the initial array-based screening data of 15 adipokines, adiponectin, leptin and resistin were selected for validation by enzyme-linked immunosorbent assay (ELISA). Correlations were determined between adipokine expression levels and measures of disease activity or lupus nephritis. The expression of adiponectin and resistin was increased in both sera and urine from LN patients, while leptin was increased in LN patient sera, compared to matched controls. Serum resistin, but not urine resistin, was correlated with measures of renal dysfunction in LN. Serum resistin expression may be useful as a marker of renal dysfunction in patients with LN, although longitudinal studies are warranted. Further studies are necessary to determine if resistin has functional consequences in LN.

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death–independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8–deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type–specific manner and acts uniquely in DCs to maintain tolerance.

Summary Systemic lupus erythematosus (SLE) is a polygenic autoimmune disease characterized by the production of anti-nuclear autoantibodies that lead to subsequent end organ damage. Previous array-based studies in patients with SLE have shown that high immunoglobulin (Ig)G anti-nuclear autoantibody reactivity was associated with severe renal lupus, whereas IgM polyreactivity was associated with less severe disease. To ascertain how different murine lupus strains recapitulate these different autoantibody profiles seen in patients, serum from New Zealand black (NZB)/NZ white (W) F1, Murphy Roths large (MRL)/lpr, NZ mixed (M)2410 and BXSB strains were compared using a comprehensive array-based screen. The array results were verified using enzyme-linked immunosorbent assays (ELISAs). Serum from MRL/lpr mice exhibited high levels of IgG anti-nuclear antibodies as well as anti-glomerular antibodies and variable levels of antibodies to myosin, Matrigel and thyroglobulin. Elevated anti-nuclear IgG antibodies were associated with severe nephritis in this strain. In contrast, NZM2410 mice exhibited lower IgG autoantibody levels with less severe nephritis but a significantly higher polyreactive IgM autoantibody profile. ELISA analysis confirmed these results. The NZB/NZW F1 and BXSB strains exhibited an intermediate serological profile. Hence, just as in patients with SLE, whereas strong IgG reactivity to nuclear antigens is associated with severe renal disease, a polyreactive IgM seroprofile is also less ominous in murine lupus.

The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway, and its role in mediating lymphoproliferation has been extensively examined. The present study was undertaken to investigate the impact of myeloid cell-specific loss of Fas.Mice with Fas flanked by loxP sites (Fas(flox/flox) ) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (Cre(LysM) ), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for Cre(LysM) Fas(flox/flox) mice was verified by polymerase chain reaction and flow cytometric analysis. Flow cytometric analysis was also used to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood, and spleen. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure serum cytokine/chemokine and immunoglobulin levels. Renal damage or dysfunction was examined by immunohistochemical and immunofluorescence analysis.Cre(LysM) Fas(flox/flox) mice exhibited a systemic lupus erythematosus (SLE)-like disease that included leukocytosis, splenomegaly, hypergammaglobulinemia, antinuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1(low) and Gr-1(intermediate) blood monocytes and splenic macrophages and, in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in Cre(LysM) Fas(flox/flox) mice.Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice, reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor of systemic autoimmunity.

Author(s): Peng, Ai; Wu, Tianfu; Zeng, Caihong; Rakheja, Dinesh; Zhu, Jiankun; Ye, Ting; Hutcheson, Jack; Vaziri, Nosratola D.; Liu, Zhihong; Mohan, Chandra; Zhou, Xin J. | Abstract: BackgroundDysregulaiton of phosphate homeostasis as occurs in chronic kidney disease is associated with cardiovascular complications. It has been suggested that both hyperphosphatemia and hypophosphatemia can cause cardiovascular disease. The molecular mechanisms by which high or low serum phosphate levels adversely affect cardiovascular function are poorly understood. The purpose of this study was to explore the mechanisms of endothelial dysfunction in the presence of non-physiologic phosphate levels.Methodology/Principal FindingsWe studied the effects of simulated hyper- and hypophosphatemia in human umbilical vein endothelial cells in vitro. We found both simulated hyperphosphatemia and hypophosphatemia decrease eNOS expression and NO production. This was associated with reduced intracellular calcium, increased protein kinase C β2 (PKCβ2), reduced cell viability, and increased apoptosis. While simulated hyperphosphatemia was associated with decreased Akt/p-Akt, Bcl-xl/Bax ratios, NFkB-p65 and p-Erk abundance, simulated hypophosphatemia was associated with increased Akt/p-Akt and Bcl-xl/Bax ratios and p-Mek, p38, and p-p38 abundance.Conclusions/SignificanceThis is the first demonstration of endothelial dysfunction with hypophosphatemia. Our data suggests that both hyperphosphatemia and hypophosphatemia decrease eNOS activity via reduced intracellular calcium and increased PKCβ2. Hyperphosphatemia also appears to reduce eNOS transcription via reduced signaling through PI3K/Akt/NF-kB and MAPK/NF-kB pathways. On the other hand, hypophosphatemia appears to activate these pathways. Our data provides the basis for further studies to elucidate the relationship between altered phosphate homeostasis and cardiovascular disease. As a corollary, our data suggests that the level of phosphate in the culture media, if not in the physiologic range, may inadvertently affect experimental results.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by a defect in immune tolerance and exacerbated by both the innate and adaptive arms of the immune response. SLE-associated immune hyperactivity can be detected systemically as elevations in levels of cytokines along with their upregulated receptors expressed by hematopoietic cells. Importantly, increased levels of cytokines and their receptors can be observed in target organs, and it is clear that they have important roles in disease pathogenesis. Recent therapeutic strategies have focused on proximal cytokines, such as interferon-α, interleukin (IL)-1, IL-6, and tumor necrosis factor as a result of the efficacious use of biologic agents for intervention in rheumatoid arthritis and other autoimmune diseases. Despite the recent advances in understanding the cytokine networks involved in autoimmune diseases and more specifically in SLE, the diagnosis and prognosis of lupus remain a challenge. Lupus is heterogeneous and unpredictable; moreover, the frequency and severity of flares can be difficult to determine and treat. A better understanding of the regulation of expression of key cytokines and their receptors can likely provide important clues to the pathogenic mechanisms underlying specific forms of SLE, and pave the way toward more effective therapeutics.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

Rheumatoid arthritis (RA) is a debilitating disease, resulting in the destruction of bone and cartilage, and in the permanent disfigurement of joints. Although the precise cause of RA is currently unresolved, it has become clear that the damaging effects are a result of the toxic milieu caused by an influx of inflammatory cells and the resulting heightened proinflammatory state within the joint. As the amount of literature suggesting that this preponderance of cells is a result of decreased local apoptosis in the joint continues to increase, in this review, we describe how Bcl-2 family pro-apoptotic BH3-only proteins, particularly Bim and Bid, could act to protect against the development of the disease. We also suggest a role for BH3-mimetic drugs as potential therapeutics in the treatment of RA.

Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1beta in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis.

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1β and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.

Objective—Fas ligand (FasL) can induce apoptosis in cells bearing the Fas receptor. The role of FasL in the vasculature with regard to atherosclerosis is controversial. This study examined the function of endothelial FasL during atherosclerosis.Methods and Results—Transgenic (Tg) mice that specifically overexpress different levels of FasL on vascular endothelial cells were crossed into the apolipoprotein E–knockout background (ApoE-KO) to generate ApoE-KO/FasL–Tg mice. Although plasma cholesterol and triglyceride levels were not different between ApoE-KO/FasL–Tg mice and ApoE-KO mice after 12 weeks of a high-fat diet, overexpression of the FasL transgene significantly reduced atherosclerotic lesion area in aortae by 49%. The reduction of atherosclerotic lesion area was more pronounced in thoracic and abdominal aortae than in the aortic arch, and a 34% reduction in lesion area was observed in aortic root sections from the ApoE-KO/FasL–Tg group compared with the ApoE-KO group. Immunostaining revealed significant decreases in both macrophage and CD8 T-cell accumulation in lesions of ApoE-KO/FasL–Tg mice. ApoE-KO/FasL–Tg mice that express lower levels of endothelial FasL also displayed reduced lesion size, but this reduction was statistically significant at the aortic arch only.Conclusions—Overexpression of endothelial FasL is antiinflammatory and inhibits atherosclerosis under hypercholesterolemic conditions.

Introduction Systemic lupus erythematosus is a chronic autoimmune disease characterized by an abundance of autoantibodies against nuclear antigens. Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation. Recently, selective inhibition of Btk by PCI-32765 has shown promise in limiting activity of multiple cells types in various models of cancer and autoimmunity. The aim of this study was to determine the effect of Btk inhibition by PCI-32765 on the development of lupus in lupus-prone B6.Sle1 and B6.Sle1.Sle3 mice. Methods B6.Sle1 or B6.Sle1.Sle3 mice received drinking water containing either the Btk inhibitor PCI-32765 or vehicle for 56 days. Following treatment, mice were examined for clinical and pathological characteristics of lupus. The effect of PCI-32765 on specific cell types was also investigated. Results In this study, we report that Btk inhibition dampens humoral autoimmunity in B6.Sle1 monocongenic mice. Moreover, in B6.Sle1.Sle3 bicongenic mice that are prone to severe lupus, Btk inhibition also dampens humoral and cellular autoimmunity, as well as lupus nephritis. Conclusions These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.

Alterations in the stoichiometric balance between members of Bcl-2 and Fas apoptotic pathway could lead to the pathogenesis of systemic lupus erythematosus (SLE). We showed that patients with SLE displayed increased expression in antiapoptotic members of the Bcl-2 and Fas apoptotic pathways in isolated mononuclear cells. Further, mice (Bcl2l11(-/-)Fas(lpr/lpr)) lacking the Bcl-2 pro-apoptotic member, Bim (Bcl2l11(-/-)) and and with an lpr mutation in the gene encoding Fas (Fas(lpr/lpr)) developed severe SLE-like disease by 16 weeks of age unlike Bcl2l11(-/-) or Fas(lpr/lpr) mice. Bcl2l11(-/-)Fas(lpr/lpr) antigen-presenting cells (APCs) were markedly activated, and their numbers were increased in lymphoid tissues and in kidneys, yet numerous TUNEL-positive cells were observed in glomeruli of Bcl2l11(-/-)Fas(lpr/lpr) mice. These data demonstrate that dysregulation of the Bcl-2 or Fas pathways can alter the function of APCs, thereby leading to SLE pathogenesis.