Curcumin Attenuates Both Acute and Chronic Immune Nephritis.

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.

Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

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.

Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88-mediated dendritic cell activation.

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.

RB tumor suppressive function in response to xenobiotic hepatocarcinogens.

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.

Retinoblastoma protein potentiates the innate immune response in hepatocytes: significance for hepatocellular carcinoma.

UNLABELLED: 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.

Adipokines influence the inflammatory balance in autoimmunity.

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.

25% HUMAN SERUM ALBUMIN IMPROVES HEMODYNAMICS AND PREVENTS THE NEED FOR NEARLY ALL PREHOSPITAL RESUSCITATION IN A RAT ( RATTUS NORVEGICUS ) MODEL OF TRAUMA AND HEMORRHAGE.

ABSTRACT: Combat casualty care can be complicated by transport times exceeding the "golden hour," with intervention and resuscitation limited to what the medic can carry. Pharmaceutical albumin comes highly saturated with nonesterified fatty acids (NEFAs). We recently showed that treatment with 25% bovine serum albumin (BSA) loaded with oleic acid, but not NEFA-free BSA, improved survival for hours after severe hemorrhage and often eliminated the need for resuscitation in rats. However, it was unknown whether pharmaceutical albumin, derived from human sources and loaded with caprylic acid (CA), would have the same benefits. We compared adjunct treatment with oleic acid-saturated BSA, CA-saturated BSA, pharmaceutical human serum albumin, or a no-albumin control in a similar rat hemorrhagic shock model to determine whether the three NEFA-albumin groups provided the same benefits relative to control. We found almost no significant differences among the NEFA-albumin groups in any measure. Mortality in controls was too low to allow for detection of improvement in survival, but NEFA-albumin groups had significantly improved hemodynamics, lactate clearance, and greatly reduced fluid requirements compared with controls. Contrary to expectations of "dehydration," 25% albumins shifted little additional fluid into the vasculature. Rather, they restored protein to the autotransfusion fluid. Nonesterified fatty acids-albumin did not worsen lung permeability, but we observed a loss of circulating protein suggesting it may have increased overall vascular permeability. Our findings suggest that, though imperfect, 25% human serum albumin could be a solution for resuscitation in austere conditions requiring prolonged field care.

iTRAQ-based mass spectrometry screen to identify serum biomarkers in systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder with no reliable serum biomarkers currently available other than autoantibodies. METHODS: In the present study, isobaric tags for relative and absolute quantitation-based mass spectrometry was used to screen the sera of patients with SLE to uncover potential disease biomarkers. RESULTS: 85 common proteins were identified, with 16 being elevated (≥1.3) and 23 being decreased (≤0.7) in SLE. Of the 16 elevated proteins, serum alpha-1-microglobulin/bikunin precursor (AMBP), zinc alpha-2 glycoprotein (AZGP) and retinol-binding protein 4 (RBP4) were validated in independent cross-sectional cohorts (Cohort I, N=52; Cohort II, N=117) using an orthogonal platform, ELISA. Serum AMBP, AZGP and RBP4 were validated to be significantly elevated in both patients with inactive SLE and patients with active SLE compared with healthy controls (HCs) (p<0.05, fold change >2.5) in Cohort I. All three proteins exhibited good discriminatory power for distinguishing active SLE and inactive SLE (area under the curve=0.82-0.96), from HCs. Serum AMBP exhibited the largest fold change in active SLE (5.96) compared with HCs and correlated with renal disease activity. The elevation in serum AMBP was validated in a second cohort of patients with SLE of different ethnic origins, correlating with serum creatinine (r=0.60, p<0.001). CONCLUSION: Since serum AMBP is validated to be elevated in SLE and correlated with renal disease, the clinical utility of this novel biomarker warrants further analysis in longitudinal cohorts of patients with lupus and lupus nephritis.

Requirement of myeloid cell-specific Fas expression for prevention of systemic autoimmunity in mice.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Peritoneal catheter implantation elicits IL-10-producing immune-suppressor macrophages through a MyD88-dependent pathway.

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.

Combined loss of proapoptotic genes Bak or Bax with Bim synergizes to cause defects in hematopoiesis and in thymocyte apoptosis.

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 CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages.

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.

Loss of Bim results in abnormal accumulation of mature CD4-CD8-CD44-CD25- thymocytes.

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 TCRbeta. Further, the CD4(-)CD8(-)CD25(-)CD44(-)CD3(+)TCRbeta(+) 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.

Biological specificity of CDK4/6 inhibitors: dose response relationship, in vivo signaling, and composite response signature.

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.

Bim suppresses the development of SLE by limiting myeloid inflammatory responses.

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-ß) 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.

Immunologic and Metabolic Features of Pancreatic Ductal Adenocarcinoma Define Prognostic Subtypes of Disease.

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.

Endothelial overexpression of Fas ligand decreases atherosclerosis in apolipoprotein E-deficient mice.

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.

The RB tumor suppressor at the intersection of proliferation and immunity: relevance to disease immune evasion and immunotherapy.

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.

Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

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: Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/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. Cre (LysM) Casp8 (fl/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 Ly6C(high)CD11b(+)F4/80(+) splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in Cre (LysM) Casp8 (fl/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.

Modulating proximal cell signaling by targeting Btk ameliorates humoral autoimmunity and end-organ disease in murine lupus.

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.