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Background Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage. Results Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17–20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands − 2, − 3, − 5 and − 11) as compared to lavage. Conclusions Synovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Fusion genes including NPM–ALK can promote T-cell transformation, but the signals required to drive a healthy T cell to become malignant remain undefined. In this study, we introduce NPM–ALK into primary human T cells and demonstrate induction of the epithelial-to-mesenchymal transition (EMT) program, attenuation of most T-cell effector programs, reemergence of an immature epigenomic profile, and dynamic regulation of c-Myc, E2F, and PI3K/mTOR signaling pathways early during transformation. A mutant of NPM–ALK failed to bind several signaling complexes including GRB2/SOS, SHC1, SHC4, and UBASH3B and was unable to transform T cells. Finally, T-cell receptor (TCR)–generated signals were required to achieve T-cell transformation, explaining how healthy individuals can harbor T cells with NPM–ALK translocations. These findings describe the fundamental mechanisms of NPM–ALK-mediated oncogenesis and may serve as a model to better understand factors that regulate tumor formation. Significance: This investigation into malignant transformation of T cells uncovers a requirement for TCR triggering, elucidates integral signaling complexes nucleated by NPM–ALK, and delineates dynamic transcriptional changes as a T cell transforms. See related commentary by Spasevska and Myklebust, p. 3160

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient–derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF–MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

A 20-year-old Quarter Horse gelding was presented with severe right forelimb lameness (5/5 AAEP Lameness Scale) due to a tear of the superficial digital flexor muscle which was diagnosed via palpation of swelling and ultrasonography revealing major muscle fiber disruption and hematoma formation. When traditional systemic therapy (non-Steroidal anti-inflammatories) did not restore clinically acceptable comfort and the risk of supporting limb laminitis became a reasonable concern, a cervical epidural catheter was placed between the first and second cervical vertebrae in the standing, sedated patient using ultrasound guidance. The gelding was treated with epidural morphine (0.1 mg/kg every 24 h then decreased to 0.05 mg/kg every 12 h) and was pain-scored serially following treatment. Spinal analgesia was provided for 3 days. Pain scores significantly decreased following each treatment with morphine, and the gelding was successfully managed through the acutely painful period without any adverse effects associated with the C1-C2 epidural catheter placement technique, the epidural morphine, or contralateral limb laminitis. At the 2-month follow-up, the gelding was walking sound with no complications seen at the catheter insertion site. In this case, spinal analgesia using epidural morphine administered via a cervical epidural catheter was an effective and technically achievable option for pain management associated with severe forelimb muscle injury in a horse.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the stromal component in the tumor microenvironment (TME). Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, their roles and potential mechanisms by which they may contribute to immune suppression in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining the TCGA dataset. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. To elucidate additional immunosuppressive mechanisms of CAF-educated MDSCs, we performed proteomic profiling and identified the NADPH oxidase 2 (NOX2) complex to be highly enriched in MDSCs. Pharmacologic inhibition of NOX2 activity in CAF-induced MDSCs restored CD8+ T-cell proliferation. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrates that NOX2 inhibition abrogates the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment. Citation Format: Handan Xiang, Carlo Ramil, Josephine Hai, Chunsheng Zhang, Huijun Wang, Amanda A. Watkins, Roshi Afshar, Peter Georgiev, Xuelei Song, Dongyu Sun, Andrey Loboda, Yanlin Jia, Lily Y Moy, An Chi, Philip E. Brandish. Cancer-associated fibroblasts promote immunosuppression by inducing NOX2-expressing monocytic MDSCs in lung squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A98.

Objective Polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased risk of developing rheumatoid arthritis (RA). This study was undertaken to determine the role of IRF5 in a mouse model of arthritis development. Methods K/BxN serum–transfer arthritis was induced in mice deficient in IRF5, or lacking IRF5 only in myeloid cells, and arthritis severity was evaluated. K/BxN arthritis was also induced in mice deficient in TRIF, Toll-like receptor 2 (TLR2), TLR3, TLR4, and TLR7 to determine the pathways through which IRF5 might promote arthritis. In vitro studies were performed to determine the role of IRF5 in interleukin-1 (IL-1) receptor and TLR signaling. Results Arthritis severity was reduced in IRF5-deficient, TRIF-deficient, TLR3-deficient, and TLR7-deficient mice. The expression of multiple genes regulating neutrophil recruitment or function and bioactive IL-1β formation was reduced in the joints during active arthritis in IRF5-deficient mice. In vitro studies showed that TLR7 and the TRIF-dependent TLR3 pathway induce proinflammatory cytokine production in disease-relevant cell types in an IRF5-dependent manner. Conclusion Our findings indicate that IRF5 contributes to disease pathogenesis in inflammatory arthritis. This is likely due at least in part to the role of IRF5 in mediating proinflammatory cytokine production downstream of TLR7 and TLR3. Since TLR7 and TLR3 are both RNA-sensing TLRs, this suggests that endogenous RNA ligands present in the inflamed joint promote arthritis development. These findings may be relevant to human RA, since RNA capable of activating TLR7 and TLR3 is present in synovial fluid and TLR7 and TLR3 are up-regulated in the joints of RA patients.

A subset of chronic lymphocytic leukemia (CLL) B cell receptors (BCRs) interact with antigens expressed on apoptotic cells, suggesting that CLL BCRs have the potential to internalize apoptotic cell RNA or DNA-containing fragments with resultant activation of TLR7 or TLR9, respectively. By blocking cAMP degradation, type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitors activate cAMP-mediated signaling and induce apoptosis in CLL cells. Here we show that autologous irradiated leukemic cells induce proliferation in CLL cells and that such proliferation is blocked by a TLR7/8/9 inhibitor, by DNase and by the PDE4 inhibitor rolipram. Rolipram also inhibited CLL cell proliferation induced by synthetic TLR7 and TLR9 agonists, as well as TLR agonist-induced costimulatory molecule expression and TNF-α (but not IL-6 or IL-10) production. While treatment with a TLR9 agonist protected immunoglobulin heavy chain variable region (IGHV) unmutated, but not mutated, CLL cells from apoptosis, PDE4 inhibitors augmented apoptosis in both subtypes, suggesting that cAMP-mediated signaling may abrogate a TLR9-mediated survival signal in prognostically unfavorable IGHV-unmutated CLL cells. Rolipram inhibited both TLR7/8 and TLR9-induced IRF5 and NF-κB p65 nuclear translocation. PDE4 inhibitors also blocked TLR signaling in normal human immune cells. In peripheral blood whole mononuclear cells (PBMC) and CD14-positive monocytes, PDE4 inhibitors blocked IFN-α or TNF-α (but not IL-6) production, respectively, following stimulation with synthetic TLR agonists or RNA-containing immune complexes. These results suggest that PDE4 inhibitors may be of clinical utility in CLL or autoimmune diseases that are driven by TLR-mediated signaling.

Background: Breastfeeding competencies are not standardized in healthcare education for any of the health professions. A few continuing education/professional development programs have been implemented, but research regarding the efficacy of these programs is scarce. Research aim: After a 45-hour lactation course, (a) Does breastfeeding knowledge increase? (b) Do beliefs and attitudes about infant feeding improve? (c) Does perceived behavioral control over performance of evidence-based lactation support practices increase? and (d) Do intentions to carry out evidence-based lactation support practices increase? Methods: A nonexperimental pretest–posttest self-report survey design was conducted with a nonprobability sample of participants ( N = 71) in a lactation course. Theory of Planned Behavior variables were measured and a before–after course analysis was completed. Results: Significantly higher scores were found on the posttests for knowledge, beliefs about breastfeeding scale, and the perceived behavioral control scale. Participants’ self-efficacy increased after the course; their beliefs about social norms and their ability to effect change in their workplaces did not change significantly. Participants’ intention to perform actions that are consistent with the evidence-based breastfeeding supportive behaviors increased significantly. Positive beliefs about formula feeding significantly increased; this was unexpected. Conclusion: The Theory of Planned Behavior provided a useful approach for examining more meaningful learning outcomes than the traditional knowledge and/or satisfaction outcomes. This study was the first to suggest that more meaningful learning outcomes are needed to evaluate lactation programs. However, it is not enough to educate healthcare providers in evidence-based practice; the places they practice must have the infrastructure to support evidence-based practice.

Previous research on sensitization in Aplysia was based entirely on unnatural noxious stimuli, usually electric shock, until our laboratory found that a natural noxious stimulus, a single sublethal lobster attack, causes short-term sensitization. We here extend that finding by demonstrating that multiple lobster attacks induce long-term sensitization (≥24 h) as well as similar, although not identical, neuronal correlates as observed after electric shock. Together these findings establish long- and short-term sensitization caused by sublethal predator attack as a natural equivalent to sensitization caused by artificial stimuli.

The importance of breastmilk as a primary preventative intervention is widely known and understood by most healthcare providers. The actions or non-actions that heathcare providers take toward promoting and supporting breastfeeding families make a difference in the success and duration of breastfeeding. Recognizing this relationship, the World Health Organization developed the International Code of Marketing of Breast-milk Substitutes (the Code), which defines best practices in breastfeeding promotion, including physicians' offices. The pediatric practices' waiting rooms are often a family's first experience with pediatric care. The specific aims of this study were to describe (1) Code compliance, (2) the demographic factors affecting the Code compliance, and (3) the amount and type of breastfeeding-supportive materials available in the pediatricians' waiting rooms. An observational cross-sectional design was used to collect data from 163 (82%) of the pediatric practices in Maricopa County, Arizona. None of the 100 waiting rooms that had any materials displayed (61%) was found to be completely Code compliant, with 81 of the offices having formula-promotional materials readily available. Waiting rooms in higher income areas offered more non-Code-compliant materials and gifts. Breastfeeding support information and materials were lacking in all but 18 (18%) offices. A positive relationship (t97=-2.31, p=0.02) occurred between the presence of breastfeeding educational materials and higher income areas. We were able to uncover some practice-related patterns that impact families and potentially undermine breastfeeding success. To move current practices toward breastfeeding-friendly physicians' offices, change is needed.

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

In Hong Kong during the severe acute respiratory syndrome outbreak of 2003, sustained uncertainty caused daily stress for residents for > 3 months. Expectant women experienced unexpected social disruption and isolation within their day-to-day life that have not been described in their own voice. The purpose of this study was to describe the experiences of women who became mothers during the outbreak and the ways in which these experiences impacted their early post-partum mothering. A phenomenological research design was chosen. The participants' responses then led the interview process. As the women's experiences had many similarities, saturation was reached after eight interviews. Four themes emerged: living with uncertainty, intense vigilance, isolation, and disrupted expectations. The participants spoke of disrupted daily routines as they tried to eliminate their risk of contracting this disease, including relationship difficulties with their spouse. None of the women had the birth experience they had hoped for because of changes in hospital practices.

Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcγRIIB−/−Yaa and FcγRIIB−/− lupus models. In contrast to IRF5-sufficient FcγRIIB−/−Yaa mice, IRF5-deficient FcγRIIB−/−Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-α, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcγRIIB−/−Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcγRIIB−/−Yaa mice, type I IFN receptor subunit 1-deficient FcγRIIB−/−Yaa mice maintained a substantial level of residual disease. Furthermore, in FcγRIIB−/− mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcγRIIB−/−Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

Premature atherosclerosis is a severe complication of lupus and other systemic autoimmune disorders. Gain-of-function polymorphisms in IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing lupus, and IRF5 deficiency in lupus mouse models ameliorates disease. However, whether IRF5 deficiency also protects against atherosclerosis development in lupus is not known. In this study, we addressed this question using the gld.apoE−/− mouse model. IRF5 deficiency markedly reduced lupus disease severity. Unexpectedly, despite the reduction in systemic immune activation, IRF5-deficient mice developed increased atherosclerosis and also exhibited metabolic dysregulation characterized by hyperlipidemia, increased adiposity, and insulin resistance. Levels of the atheroprotective cytokine IL-10 were reduced in aortae of IRF5-deficient mice, and in vitro studies demonstrated that IRF5 is required for IL-10 production downstream of TLR7 and TLR9 signaling in multiple immune cell types. Chimera studies showed that IRF5 deficiency in bone marrow–derived cells prevents lupus development and contributes in part to the increased atherosclerosis. Notably, IRF5 deficiency in non–bone marrow–derived cells also contributes to the increased atherosclerosis through the generation of hyperlipidemia and increased adiposity. Together, our results reveal a protective role for IRF5 in lupus-associated atherosclerosis that is mediated through the effects of IRF5 in both immune and nonimmune cells. These findings have implications for the proposed targeting of IRF5 in the treatment of autoimmune disease as global IRF5 inhibition may exacerbate cardiovascular disease in these patients.

Interferon regulatory factor 5 (IRF5) polymorphisms are strongly associated with an increased risk of developing the autoimmune disease systemic lupus erythematosus. In mouse lupus models, IRF5-deficiency was shown to reduce disease severity consistent with an important role for IRF5 in disease pathogenesis. However these mouse studies were confounded by the recent demonstration that the IRF5 knockout mouse line contained a loss-of-function mutation in the dedicator of cytokinesis 2 (DOCK2) gene. As DOCK2 regulates lymphocyte trafficking and Toll-like receptor signaling, this raised the possibility that some of the protective effects attributed to IRF5 deficiency in the mouse lupus models may instead have been due to DOCK2 deficiency. We have therefore here evaluated the effect of IRF5-deficiency in the MRL/lpr mouse lupus model in the absence of the DOCK2 mutation. We find that IRF5-deficient (IRF5−/−) MRL/lpr mice develop much less severe disease than their IRF5-sufficient (IRF5+/+) littermates. Despite markedly lower serum levels of anti-nuclear autoantibodies and reduced total splenocyte and CD4+ T cell numbers, IRF5−/− MRL/lpr mice have similar numbers of all splenic B cell subsets compared to IRF5+/+ MRL/lpr mice, suggesting that IRF5 is not involved in B cell development up to the mature B cell stage. However, IRF5−/− MRL/lpr mice have greatly reduced numbers of spleen plasmablasts and bone marrow plasma cells. Serum levels of B lymphocyte stimulator (BLyS) were markedly elevated in the MRL/lpr mice but no effect of IRF5 on serum BLyS levels was seen. Overall our data demonstrate that IRF5 contributes to disease pathogenesis in the MRL/lpr lupus model and that this is due, at least in part, to the role of IRF5 in plasma cell formation. Our data also suggest that combined therapy targeting both IRF5 and BLyS might be a particularly effective therapeutic approach in lupus.

In this study, we compared human immunodeficiency virus (HIV) type 1-specific proliferative responses with HIV-1-induced intracellular cytokine production in a cohort of clinically nonprogressing patients and individuals with progressive HIV-1 infection. We found strong HIV-1-specific proliferative responses in the clinical nonprogressor cohort that correlated with significant numbers of circulating HIV-1-specific CD4(+) T cells. In contrast, HIV-1-specific proliferative responses were absent in most individuals with progressive HIV-1 infection, even though interferon-gamma-producing HIV-1-specific CD4(+) T cells were detectable by flow cytometry. The implication of these data is that the important dysfunction seen in most HIV-positive patients from very early in disease may be an inability of HIV-1-specific CD4(+) memory T cells to proliferate in response to HIV antigens rather than an absolute loss of circulating virus-specific CD4(+) T cells.

PURPOSE. The purpose of this synthesis was to review intervention studies that focused on increasing the breastfeeding knowledge, self-confidence, and supportive behaviors of healthcare professionals. DESIGN AND METHODS. Fourteen articles were identified through database searches as the basis for this review. RESULTS. Breastfeeding education can be effective in increasing the knowledge and confidence of nurses. Duration and exclusivity rates for breastfeeding may also be improved. PRACTICE IMPLICATIONS. Improving nurses' knowledge of breastfeeding is a modifiable factor that is important in supporting a mother in her decision to breastfeed.

Exacerbation of disease in systemic lupus erythematosus (SLE) is associated with bacterial infection. In conventional dendritic cells (cDCs), the TLR4 ligand bacterial LPS induces IFN-β gene expression but does not induce IFN-α. We hypothesized that when cDCs are primed by cytokines, as may frequently be the case in SLE, LPS would then induce the production of IFN-α, a cytokine believed to be important in lupus pathogenesis. In this study we show that mouse cDCs and human monocytes produce abundant IFN-α following TLR4 engagement whether the cells have been pretreated either with IFN-β or with a supernatant from DCs activated by RNA-containing immune complexes from lupus patients. This TLR4-induced IFN-α induction is mediated by both an initial TRIF-dependent pathway and a subsequent MyD88-dependent pathway, in contrast to TLR3-induced IFN-α production, which is entirely TRIF-dependent. There is also a distinct requirement for IFN regulatory factors (IRFs), with LPS-induced IFN-α induction being entirely IRF7- and partially IRF5-dependent, in contrast to LPS -induced IFN-β gene induction which is known to be IRF3-dependent but largely IRF7-independent. This data demonstrates a novel pathway for IFN-α production by cDCs and provides one possible explanation for how bacterial infection might precipitate disease flares in SLE.

Abstract 1774 A growing body of literature supports the concept that at least a subset of chronic lymphocytic leukemia (CLL) B cell-receptors (BCRs) are reactive with antigens exposed on apoptotic cells. Work done in the autoimmunity field suggests that through their BCRs, autoreactive B cells can internalize RNA and DNA-containing autoantigens, thereby activating endosomal Toll-like receptor (TLR) 7 and TLR9, respectively, with subsequent TLR-mediated signaling, proliferation and cytokine secretion. cAMP signaling modulates inflammatory signaling in immune system cells and our prior studies have shown that PKA, EPAC and CREB-mediated cAMP signaling can be potently induced in CLL cells with inhibitors of the cyclic nucleotide phosphodiesterase (PDE) PDE4 family in the absence of additional exogenous stimulators of adenylate cyclase. We therefore examined the ability of PDE4 inhibitor-mediated cAMP signaling to regulate TLR7 and 9-mediated signaling in CLL cells. Treatment of CLL cells with the TLR7 and 9 agonists R848 and CpG-B oligonucleotide, respectively, induced IRAK1 degradation and activation of three signaling pathways known to be downstream of TLR7 and 9: the transcription factors NFkB and IRF5 as well as the MAP kinase JNK. The prototypic PDE4 inhibitor rolipram inhibited R848-induced TLR7 and CpG-B-induced TLR9 signaling in CLL cells as judged by Western analysis and immunofluorescent studies of NFkB and IRF5 nuclear translocation as well as JNK phosphorylation. Rolipram also blocked R848 and CpG-B-induced CLL proliferation and TNF secretion. In contrast, rolipram augmented R848 and CpG-B-induced CLL IL-10 synthesis, in keeping with prior studies documenting cAMP response elements in the IL-10 promoter. To test whether RNA and/or DNA antigens exposed in apoptotic cells might induce proliferation in CLL cells through BCR-mediated internalization and subsequent activation of TLR7 and TLR9, we irradiated CLL cells and added them in a graded fashion to live autologous CLL cells. Such exposure to irradiated cells undergoing apoptosis drove CLL proliferation in a manner that was sensitive to TLR7 and TLR9 antagonists, IRAK4 kinase inhibitors and the PDE4 inhibitor rolipram. Our work supports the concept that BCR and TLR-driven responses to apoptotic RNA and DNA-containing complexes may drive proliferation in CLL cells and that PDE4 inhibitors and other agents that target TLR signaling may prove to be effective therapeutic agents in this disease. Disclosures: No relevant conflicts of interest to declare.