Elevated Pro-inflammatory Cytokine Production Research Articles

Sex differences in inflammation correlated with estrogen and estrogen receptor-β levels in azoxymethane/dextran sodium sulfate-induced colitis-associated colorectal cancer mice

Colorectal cancer (CRC) is a type of cancer that develops in the colon or rectum and is the second leading cause of cancer-related death worldwide. Several epidemiology studies have identified a significant sexual dimorphism in CRC, with women exhibiting a lower incidence rate and delayed onset compared to men. This study aims to investigate the sexual dimorphism in the inflammatory response in colitis-associated CRC and its relationship with estrogen and estrogen receptors. An azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model was used to induce colitis-associated CRC. Five-week-old male and female mice were randomly assigned into either the control group or the AOM/DSS CRC group, with 10 mice in each group. Colitis-associated CRC was induced by injecting AOM (10 mg/kg) and administering two-cycles of DSS treatment in the drinking water. The results revealed a significant decrease in colon length exclusively in the female group, indicating more severe colonic inflammation (P < 0.01). A significant interaction was identified between sex and AOM/DSS treatment in the female AOM/DSS group, with higher visceral fat weight compared to their male counterparts (P < 0.05). The female AOM/DSS group also exhibited elevated production of M1 macrophage-related pro-inflammatory cytokines, suggesting increased tumor-associated macrophage activity. Surprisingly, the male AOM/DSS group showed a marked increase in serum estradiol levels, while the female AOM/DSS group exhibited a decrease compared to the normal control group. Additionally, a notable upregulation of both estrogen receptor α and estrogen receptor β expression was observed in the colon tissues of the AOM/DSS groups compared to the normal control groups, with estrogen receptor β expression being particularly pronounced in females. Taken together, our findings suggest that a decline in endogenous estrogen and increased estrogen receptors potentially contribute to the pro-inflammatory response in early CRC by augmenting cytokine expressions associated with M1 macrophage polarization in females.

Epigenetic and transcriptional regulation of cytokine production by Plasmodium falciparum-exposed monocytes.

Plasmodium falciparum infection causes the most severe form of malaria, where excessive production of proinflammatory cytokines can drive the pathogenesis of the disease. Monocytes play key roles in host defense against malaria through cytokine production and phagocytosis; however, they are also implicated in pathogenesis through excessive proinflammatory cytokine production. Understanding the underlying molecular mechanisms that contribute to inflammatory cytokine production in P. falciparum-exposed monocytes is key towards developing better treatments. Here, we provide molecular evidence that histone 3 lysine 4 (H3K4) methylation is key for inflammatory cytokine production in P. falciparum-exposed monocytes. In an established in vitro system that mimics blood stage infection, elevated proinflammatory TNF and IL-6 cytokine production is correlated with increased mono- and tri-methylated H3K4 levels. Significantly, we demonstrate through utilizing a pharmacological inhibitor of H3K4 methylation that TNF and IL-6 expression can be suppressed in P. falciparum-exposed monocytes. This elucidated epigenetic regulatory mechanism, controlling inflammatory cytokine production, potentially provides new therapeutic options for future malaria treatment.

Mutant huntingtin confers cell-autonomous phenotypes on Huntington’s disease iPSC-derived microglia

Huntington’s disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Neuroinflammation and microglia have been implicated in HD pathology, however it has been unclear if mutant HTT (mHTT) expression has an adverse cell-autonomous effect on microglial function, or if they are only activated in response to the neurodegenerative brain environment in HD. To establish a human cell model of HD microglia function, we generated isogenic controls for HD patient-derived induced pluripotent stem cells (iPSC) with 109 CAG repeats (Q109). Q109 and isogenic Q22 iPSC, as well as non-isogenic Q60 and Q33 iPSC lines, were differentiated to iPSC-microglia. Our study supports a model of basal microglia dysfunction in HD leading to elevated pro-inflammatory cytokine production together with impaired phagocytosis and endocytosis capacity, in the absence of immune stimulation. These findings are consistent with early microglia activation observed in pre-manifest patients and indicate that mHTT gene expression affects microglia function in a cell-autonomous way.

T cell pro-inflammatory cytokine production in young adults with major depressive disorder

Background: Major Depressive Disorder (MDD) is increasingly prevalent in young adults and is a significant contributor to disease burden worldwide. Previously we have shown that young adults with MDD produce greater T cell mitochondrial ROS compared to their heathy non-depressed counterparts. Because evidence from rodent models of stress-related mood disorders demonstrates that increased T cell mitoROS drives excessive proinflammatory cytokine production, we next sought to determine if either CD4+ (Helper) or CD8+ (cytotoxic) T cells likewise produce increased proinflammatory cytokines in young adults with MDD. We hypothesized that there will be elevated proinflammatory cytokine production in young adults with MDD compared to their healthy non-depressed counterparts. Methods: Peripheral blood mononuclear cells were isolated by density centrifugation from 5 young adults with MDD having mild to moderate symptom severity and 9 non-depressed young healthy adults. Interleukin 2 (IL-2), Interleukin 6 (IL-6), Interferon Gamma (IFN-γ), and Tumor Necrosis Factor alpha (TNF-) production were assessed by flow cytometry. All data are expressed as mean SE and group differences were assessed by unpaired T test. Results: We found that adults with MDD exhibited a greater proportion of CD8+ Interleukin 6 (IL-6) (n= 8; 5 HA, 3 MDD) (HA = 22 3.5%, MDD = 255.6%; P= 0.004) producing cells compared to healthy. There were no significant differences in the proportions of CD8+ cells producing IFN-γ (HA = 14 1.2%, MDD = 3.1%; P= 0.31), IL-2 (HA =76 3.9%, MDD = 69 6.6%; P= 0.17), and TNF- (HA = 21 2.3%, MDD = 23.5%; P= 0.11) in those with MDD compared to controls. Additionally, there were no differences in CD4+ production of these same cytokines. We also assessed phenotype of the T cells using CD27 (central vs effector) and CD45 RA (Naïve vs TEMRA). Interestingly, a greater proportion of TNF- producing CD8+ and CD4+ cells exhibited an effector memory phenotype in adults with MDD compared to controls (CD8: HA = 25.8 2.4%, MDD = 46.8 3.3%; P= 0.0001; CD4+: HA=31 2.9%, MDD = 394.3%; P= 0.05). These data provide insight into the T cell subtypes that may be driving the inflammation associated with MDD. Conclusion: These preliminary findings suggest that young adults with MDD may exhibit a proinflammatory T cell phenotype characterized by greater IL-6 and TNF- production. NIH R21 MH123928 and R00133414 (JLG), NIH K01AG061271 and R01AG060395 (DWT). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

An inter-species amino acid variation modulates signaling strength and function of CD28 in vitro and in vivo

Abstract Animal models do not always reflect the human condition. One such example was a phase one clinical trial of a CD28 super agonist antibody (CD28SA). In mice, CD28SA treatment expanded Tregs and reduced autoimmune disease; yet when human volunteers received CD28SA, they experienced life-threatening cytokine storm and multi-organ failure. CD28 is constitutively expressed on T cells and provides the canonical co-stimulatory “signal 2” needed for T cell activation. The proline residues in the CD28 PYAP domain are required for downstream signaling and this motif is conserved across species. However, the human PYAP domain is adjacent to an additional proline, while murine PYAP is followed by an inactive alanine. Previous in vitro studies have suggested this single amino acid variation modulates downstream signaling. We have developed a “humanized” mouse with a CD28 A to P substitution at residue 210 adjacent to the PYAP domain (CD28 A210P). In vitro, CD28 A210PT cells had increased p-Akt, JunB abundance, and produced more IL-2 compared to WT T cells when stimulated in vitro. In vivo, immunization with antigen/CFA emulsion, CD28 A210Pmice display increased antibody production, suggestive of enhanced CD28-mediated Tfh activity. In addition, we assessed the response of the “humanized” CD28 mice to the CD28 super agonist antibody. After injection with CD28 super agonist, CD28 A210Pmice had significantly increased weight loss, elevated and sustained pro-inflammatory cytokine production, and less Treg expansion compared to WT mice. This study suggests interspecies variation in CD28 signaling due to differences outside of the well-studied signaling domains can alter outcomes of CD28 ligation and promote divergent outcomes when targeted in vivo.

Hepatocyte estrogen sulfotransferase inhibition protects female mice from concanavalin A–induced T cell–mediated hepatitis independent of estrogens

Autoimmune hepatitis (AIH) is a typical T cell-mediated chronic liver disease with a higher incidence in females. However, the molecular mechanism for the female predisposition is poorly understood. Estrogen sulfotransferase (Est) is a conjugating enzyme best known for its function in sulfonating and deactivating estrogens. The goal of this study is to investigate whether and how Est plays a role in the higher incidence of AIH in females. Concanavalin A (ConA) was used to induce T cell-mediated hepatitis in female mice. We first showed that Est was highly induced in the liver of ConA-treated mice. Systemic or hepatocyte-specific ablation of Est, or pharmacological inhibition of Est, protected female mice from ConA-induced hepatitis regardless of ovariectomy, suggesting the effect of Est inhibition was estrogen independent. In contrast, we found that hepatocyte-specific transgenic reconstitution of Est in the whole-body Est knockout (EstKO) mice abolished the protective phenotype. Upon the ConA challenge, EstKO mice exhibited a more robust inflammatory response with elevated production of proinflammatory cytokines and changed liver infiltration of immune cells. Mechanistically, we determined that ablation of Est led to the hepatic induction of lipocalin 2 (Lcn2), whereas ablation of Lcn2 abolished the protective phenotype of EstKO females. Our findings demonstrate that hepatocyte Est is required for the sensitivity of female mice to ConA-induced and T cell-mediated hepatitis in an estrogen-independent manner. Est ablation may have protected female mice from ConA-induced hepatitis by upregulating Lcn2. Pharmacological inhibition of Est might be a potential strategy for the treatment of AIH.

Neuroinflammatory mediators in acquired epilepsy: an update.

Epilepsy is a group of chronic neurological disorders that have diverse etiologies but are commonly characterized by spontaneous seizures and behavioral comorbidities. Although the mechanisms underlying the epileptic seizures mostly remain poorly understood and the causes often can be idiopathic, a considerable portion of cases are known as acquired epilepsy. This form of epilepsy is typically associated with prior neurological insults, which lead to the initiation and progression of epileptogenesis, eventually resulting in unprovoked seizures. A convergence of evidence in the past two decades suggests that inflammation within the brain may be a major contributing factor to acquired epileptogenesis. As evidenced in mounting preclinical and human studies, neuroinflammatory processes, such as activation and proliferation of microglia and astrocytes, elevated production of pro-inflammatory cytokines and chemokines, blood-brain barrier breakdown, and upregulation of inflammatory signaling pathways, are commonly observed after seizure-precipitating events. An increased knowledge of these neuroinflammatory processes in the epileptic brain has led to a growing list of inflammatory mediators that can be leveraged as potential targets for new therapies of epilepsy and/or biomarkers that may provide valued information for the diagnosis and prognosis of the otherwise unpredictable seizures. In this review, we mainly focus on the most recent progress in understanding the roles of these inflammatory molecules in acquired epilepsy and highlight the emerging evidence supporting their candidacy as novel molecular targets for new pharmacotherapies of acquired epilepsy and the associated behavioral deficits.

Effect of exogenous IL-37 on immune cells from a patient carrying a potential IL37 loss-of-function variant: A case study.

Chronic inflammatory or autoimmune diseases are commonly treated with immunosuppressive medication such as NSAIDs, corticosteroids, or antibodies against specific cytokines (TNF, IL-1 IL-17, IL-23, etc.) or signalling cascades (e.g. JAK-STAT inhibitors). Using sequencing data to locate genetic mutations in relevant genes allows the identification of alternative targets in a patient-tailored therapy setting. Interleukin (IL)-37 is an anti-inflammatory cytokine with broad effects on innate and adaptive immune cell function. Dysfunctional IL-37 expression or signalling is linked to various autoinflammatory disorders. The administration of recombinant IL-37 to hyperinflammatory patients that are non-responsive to standard treatment bears the potential to alleviate symptoms. In this case study, the (hyper)responsiveness of immune cell subsets was investigated in a single patient with a seronegative autoimmune disorder who carries a heterozygous stop-gain variant in IL37 (IL37 Chr2(GRCh37):g.113670640G>A NM_014439.3:c.51G>A p.(Trp17*)). As the patient has been non-responsive to blockage of TNF or IL-1 by Etanercept or Anakinra, respectively, additional in-vitro experiments were set out to elucidate whether treatment with recombinant IL-37 could normalise observed immune cell functions. Characterisation of immune cell function showed no elevated overall production of acute-phase pro-inflammatory cytokines by patient PBMCs and neutrophils at baseline or upon stimulation. T-cell responses were elevated, as was the metabolic activity and IL-1Ra production of PBMCs at baseline. The identified stop-gain variant in IL37 does not result in the absence of the protein in circulation. In line with this, treatment with recombinant IL-37 did overall not dampen immune responses with the exception of the complete suppression of IL-17. The heterozygous stop-gain variant in IL37 (IL37 NM_014439.3:c.51G>A p.(Trp17*)) is not of functional relevance as we observed no clear pro-inflammatory phenotype in immune cells of a patient carrying this variant.

Dual roles of ANGPTL4 in multiple inflammatory responses in stomatitis mice.

Stomatitis is inflammation of the oral mucosa. Angiopoietin-like protein 4 (ANGPTL4) has pleiotropic functions both anti-inflammatory and pro-inflammatory properties. In the present study, we tested whether there is a correlation between increased ANGPTL4 expression and inflammation in stomatitis mice and the mechanisms involved. In this study, the oral mucosa of mice was burned with 90% phenol and intraperitoneal injection of 5-fluorouracil to establish the model of stomatitis mice. The pathological changes of stomatitis mice were observed by H&E staining of paraffin section. The expressions of cytokines and ANGPTL4 were detected by fluorescence quantitative PCR, and the protein levels of ANGPTL4 were detected by western blot. Compared with control group, the oral mucosal structure of model mice was damaged. The expression of ANGPTL4 were significantly increased concomitantly with elevated production of anti-inflammatory cytokine (peroxisome proliferator-activated receptoralpha) and pro-inflammatory cytokines [nuclear transcription factor-kappaB, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α] in mice with stomatitis. This study suggests that ANGPTL4 may be a double-edged sword in multiple inflammatory responses in stomatitis mice.

Functional and Phenotypic Diversity of Microglia: Implication for Microglia-Based Therapies for Alzheimer's Disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and is closely associated with the accumulation of β-amyloid (Aβ) and neurofibrillary tangles (NFTs). Apart from Aβ and NFT pathologies, AD patients also exhibit a widespread microglial activation in various brain regions with elevated production of pro-inflammatory cytokines, a phenomenon known as neuroinflammation. In healthy central nervous system, microglia adopt ramified, “surveying” phenotype with compact cell bodies and elongated processes. In AD, the presence of pathogenic proteins such as extracellular Aβ plaques and hyperphosphorylated tau, induce the transformation of ramified microglia into amoeboid microglia. Ameboid microglia are highly phagocytic immune cells and actively secrete a cascade of pro-inflammatory cytokines and chemokines. However, the phagocytic ability of microglia gradually declines with age, and thus the clearance of pathogenic proteins becomes highly ineffective, leading to the accumulation of Aβ plaques and hyperphosphorylated tau in the aging brain. The accumulation of pathogenic proteins further augments the neuroinflammatory responses and sustains the activation of microglia. The excessive production of pro-inflammatory cytokines induces a massive loss of functional synapses and neurons, further worsening the disease condition of AD. More recently, the identification of a subset of microglia by transcriptomic studies, namely disease-associated microglia (DAM), the progressive transition from homeostatic microglia to DAM is TREM2-dependent and the homeostatic microglia gradually acquire the state of DAM during the disease progression of AD. Recent in-depth transcriptomic analysis identifies ApoE and Trem2 from microglia as the major risk factors for AD pathogenesis. In this review, we summarize current understandings of the functional roles of age-dependent microglial activation and neuroinflammation in the pathogenesis of AD. To this end, the exponential growth in transcriptomic data provides a solid foundation for in silico drug screening and gains further insight into the development of microglia-based therapeutic interventions for AD.

Vitamin D modulates the expression of Toll-like receptors and pro-inflammatory cytokines without affecting Chikungunya virus replication, in monocytes and macrophages

Chikungunya virus (CHIKV) is a zoonotic arthropod-borne virus that causes Chikungunya fever (CHIKF), a self-limiting disease characterized by myalgia and acute or chronic arthralgia. CHIKF pathogenesis has an important immunological component since higher levels of pro-inflammatory factors, including cytokines and chemokines, are detected in CHIKV-infected patients. In vitro studies, using monocytes and macrophages have shown that CHIKV infection promotes elevated production of pro-inflammatory cytokines and antiviral response factors. Vitamin D3 (VD3) has been described as an important modulator of immune response and as an antiviral factor for several viruses. Here, we aimed to study the effects of VD3 treatment on viral replication and pro-inflammatory response in CHIKV-infected human monocytes (VD3-Mon) and monocyte-derived macrophages differentiated in the absence (MDMs) or the presence of VD3 (VD3-MDMs). We found that VD3 treatment did not suppress CHIKV replication in either VD3-Mon or VD3-MDMs. However, the expression of VDR, CAMP and CYP24A1 mRNAs was altered by CHIKV infection. Furthermore, VD3 treatment alters TLRs mRNA expression and production of pro-inflammatory cytokines, including TNFα and CXCL8/IL8, but not IL1β and IL6, in response to CHIKV infection in both VD3-Mon and VD3-MDMs. While a significant decrease in CXCL8/IL8 production was observed in CHIKV-infected VD3-Mon, significantly higher production of CXCL8/IL8 was observed in CHIKV-infected VD3-MDM at 24 hpi. Altogether, our results suggest that vitamin D3 may play an important role in ameliorating pro-inflammatory response during CHIKV infection in human Mon, but not in MDMs. Although further studies are needed to evaluate the efficacy of VD3; nevertheless, this study provides novel insights into its benefits in modulating the inflammatory response elicited by CHIKV infection in humans.

Hematopoietic Stem and Progenitor Cells Improve Survival from Sepsis By Boosting Immunomodulatory Cells

Sepsis is a dysregulated inflammatory syndrome that accounts for as many as 20% of deaths worldwide. Elevated production of pro-inflammatory cytokines during sepsis, such as IL-1, IL-6, interferons (IFNs), and tumor necrosis factor contribute to the development of fever, vasodilation, and multiorgan failure. Novel therapies to treat sepsis are urgently needed.Hematopoietic stem and progenitor cells (HSPC) are responsible for the day-to-day production of blood and immune cells. Recent work from our group and others indicates that during emergency hematopoiesis, inflammatory signals including cytokines, chemokines, and pathogen-derived molecules direct HSPCs to differentiate into effector immune cells. While these signals are essential for a proper immune response, excessive signaling in HSPCs can be detrimental and lead to their depletion. Thus, the interactions between HSPCs and their inflammatory environment may play a deterministic role in immune responses and sepsis.We used a mouse model of Group A Streptococcus (GAS) infection to examine the role of HSPCs in pathogenic infection and sepsis. GAS is a common pathogen that can cause a plethora of diseases from mild skin infections to life-threatening necrotizing fasciitis. We infected mice with 10 6 cfu GAS by intramuscular injection, which typically results in sepsis and death within 7 days, and examined the impact of this infection on peripheral blood (PB) and bone marrow (BM) populations. In just 24 hrs after GAS infection, BM myeloid and HSPC populations are significantly depleted, with myeloid cells being heavily trafficked into circulation following increased levels of monocyte chemoattractant protein-1 (MCP-1). Lineage tracing experiments using KRT18-CreERT2:Rosa26-lox-STOP-lox-TdTomato demonstrated that endogenous HSPCs differentiate toward the myeloid lineage after GAS infection. Based on these data, we hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation resulting in a depletion that could be rescued by the infusion of new HSPCs. To test this hypothesis, we infused GAS-infected mice with 10 4 naïve HSPCs (1.7x10 7 cells per m 2) and evaluated pathogen load and overall survival. This number of HSPCs infused is very low in comparison to the current granulocyte therapies that use ~10 10 cells per m 2 cells per infusion. BM and PB analysis showed that HSPC infusion restored HSPC levels and significantly increased myeloid progenitors and circulating myeloid cells. Strikingly, HSPC infusion in GAS-infected mice significantly increased survival, with 50-75% of mice surviving as opposed to 0-10% of controls. Despite the restoration of hematopoietic populations, surprisingly, GAS-infected mice infused with HSPCs did not show a reduction in pathogen load.Given that HSPC infusion significantly increased survival without impacting pathogen clearance, we sought to determine whether infused HSPCs served an immunomodulatory role. Analysis of BM and PB did not show any changes in lymphocyte populations, suggesting that Tregs and Bregs were not strongly affected. However, BM and PB MDSC populations were severely depleted during GAS sepsis, and HSPC infusion led to a dramatic restoration of these MDSC populations. Interestingly and in accordance with MDSC numbers, the overall cytokine levels of GAS-infected mice are lower after HSPC infusion. Notably, serum levels of cytokines known to drive the symptoms of sepsis, like TNF, IL-12, MIP-1a, IL-6, and IL-1b were dampened in HSPC-rescued mice.In conclusion, while HSPC infusion did not reduce bacterial load, it conferred a significant survival advantage to GAS-infected mice. Our data showing restoration of MDSCs and lower cytokine levels after HSPC infusion suggest that HSPC infusion supports the development of immunomodulatory cells that can prevent sepsis-related hyperinflammation and death. Current work is directed at defining specific HSPC subpopulations that mediate this effect. Importantly, the rescue potential of such low numbers of infused HSPCs highlights the feasibility of this technique and its potential applications. Overall, the information gained in this project may contribute to a new therapeutic strategy to use HSPCs to fight bacterial infections and sepsis where granulocyte infusions have so far produced only mixed results. DisclosuresNo relevant conflicts of interest to declare.

Galectin-3 regulates proinflammatory cytokine function and favours Brucella abortus chronic replication in macrophages and mice.

In this study, we provide evidence that galectin-3 (Gal-3) plays an important role in Brucella abortus infection. Our results showed increased Gal-3 expression and secretion in B. abortus infected macrophages and mice. Additionally, our findings indicate that Gal-3 is dispensable for Brucella-containing vacuoles disruption, inflammasome activation and pyroptosis. On the other hand, we observed that Brucella-induced Gal-3 expression is crucial for induction of molecules associated to type I IFN signalling pathway, such as IFN-β: Interferon beta (IFN-β), C-X-C motif chemokine ligand 10 (CXCL10) and guanylate-binding proteins. Gal-3 KO macrophages showed reduced bacterial numbers compared to wild-type cells, suggesting that Gal-3 facilitates bacterial replication in vitro. Moreover, priming Gal-3 KO cells with IFN-β favoured B. abortus survival in macrophages. Additionally, we also observed that Gal-3 KO mice are more resistant to B. abortus infection and these animals showed elevated production of proinflammatory cytokines when compared to control mice. Finally, we observed an increased recruitment of macrophages, dendritic cells and neutrophils in spleens of Gal-3 KO mice compared to wild-type animals. In conclusion, this study demonstrated that Brucella-induced Gal-3 is detrimental to host and this molecule is implicated in inhibition of recruitment and activation of immune cells, which promotes B. abortus spread and aggravates the infection. TAKE AWAYS: Brucella abortus infection upregulates galectin-3 expression Galectin-3 regulates guanylate-binding proteins expression but is not required for Brucella-containing vacuole disruption Galectin-3 modulates proinflammatory cytokine production during bacterial infection Galectin-3 favours Brucella replication.

TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents &amp; Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

LB920 IQGAP3 is an important mediator of the psoriatic phenotype of keratinocytes

Keratinocytes are immunocompetent cells important for the structural and barrier function of skin. Psoriatic keratinocytes are characterized by the enhanced proliferation and reduced differentiation rates as well as by the elevated production of proinflammatory cytokines and chemoattractants. In this research we have knocked-down a scaffold protein IQGAP3 using shRNA in HaCaT keratinocytes (HaCaT_shIQ3) in order to test the hypothesis if IQGAP3 mediates the psoriatic phenotype of keratinocytes. IQGAP interacts with cell adhesion molecules, with the cytoskeleton, with signaling molecules to regulate cell morphology, motility and kinase pathways. Earlier we have identified IQGAP3 to be overexpressed in skin of psoriatic patients and to be stimulated by the proinflammatory cytokines IL17, TNFa and IFNg. RNA-seq of the HaCaT_shIQ3 cells has shown that among the GO enriched by the downregulated genes were: positive regulation of MAPK cascade, negative regulation of inflammatory response, epidermis development, negative chemotaxis, cell adhesion, keratinization, keratinocyte differentiation, intracellular signal transduction. Among the GO enriched by the upregulated genes were positive regulation of macrophage cytokine production, extracellular matrix organization, positive regulation of I-kB/NF-kB signaling, calcium transport, cell adhesion, positive regulation of inflammatory response, lipid metabolic process, positive regulation of IL6 production, NO biosynthetic process. Almost all the onthologies mentioned above are involved in psoriatic process, highlighting the importance of the IQGAP3 for this disease. Using xCELLigence real time cell analysis system we have evaluated the growth rates of HaCaT_shIQ3. Compared to the control cells, HaCaT_shIQ3 demonstrated slower growing rates, delayed wound healing and were in a lesser degree growth-stimulated by psoriatic proinflammatory cytokines. Thus we concluded IQGAP3 to be an important mediator of the psoriatic phenotype of keratinocytes.

Increased TLR4 pathway activation and cytokine imbalance led to lipopolysaccharide tolerance in monocytes from preeclamptic women.

Preeclampsia (PE) is a pregnancy syndrome characterized by a systemic inflammatory response, and endogenous activation of monocytes. This study aimed to determine whether the activation of monocytes from preeclamptic women might interfere with the response to lipopolysaccharide (LPS)-in vitro stimulation. Fifty-two preeclamptic women and 32 normotensive (NT) pregnant women were included. Monocytes from peripheral blood were cultured with or without LPS. TLR4 expression was analyzed by flow cytometry, NF-κB activity was determined in nuclear extracts and cytokines production was evaluated by ELISA. Endogenous TLR4 ligands such as Hyaluronan, HMGB1 and Hsp70 were determined in plasma. The endogenous TLR4 expression and activation of NF-κB were statistically higher in monocytes from women with PE compared to NT group. Early-onset PE showed higher TLR4 expression compared to late-onset PE. Plasma levels of Hyaluronan, HMGB1, and Hsp70, as well as endogenous production of inflammatory cytokines, were elevated whilst lower production of IL-10 was observed in the PE group. After culture with LPS, monocytes presented lower NF-κB activation, TNF-α and IL-12 production in PE groups than in the NT group. The study demonstrates endogenous activation of monocytes from preeclamptic women, accompanied by higher expression of TLR4, NF-κB activation and elevated production of pro-inflammatory cytokines. The higher plasma levels of the TLR4 ligands hyaluronan, HMGB1 and hsp70, as well as the high concentration of TNF-α endogenously produced by monocytes, could induce the LPS tolerance phenomenon in these cells. These results suggest that monocytes play an important role in the maternal excessive systemic inflammatory response in PE.

Characterization, pro-inflammatory response and cytotoxic profile of bioaerosols from urban and rural residential settings in Pune, India

Microbiota associated with airborne particulate matter (PM) is an important indicator of indoor pollution as they can be pathogenic and cause serious health threats to the exposed occupants. Present study aimed to investigate the level of culturable microbes associated with PM and their toxicological characterization in urban and rural houses of Pune city. Highest concentration of bacterial aerosols observed to be associated with PM10 size fraction in urban site (2136 ± 285 CFU/m3) whereas maximum fungal concentration has been measured in rural houses (1521 ± 302 CFU/m3). Predominantly found bacterial species were Bacillus sp., S. aureus, and Pseudomonas aeruginosa and fungal species were Aspergillus sp., Cladosporium sp., and Penicillium sp. in both urban and rural residential premises. Concentration of endotoxin measured using the kinetic Limulus Amebocyte Lysate assay exhibited that the level of endotoxin in both urban and rural sites are associated with household characteristics and the activities performed in indoor as well as outdoor. Cell free DTT assay confirmed the ability of these airborne microbes to induce the production of reactive oxygen species (ROS) varying along with the types of microorganisms. On exposure of A549 cells to airborne microbes, a significant decrease in cell viability was observed in terms of both necrosis and apoptosis pathway. Elevated production of nitric oxide (NO) and proinflammatory cytokines in epithelial cells and macrophages clearly suggest the inflammatory nature of these airborne microbes. Results derived from the present study demonstrated that the indoor air of urban and rural houses of Pune is contaminated in terms of microbial load. Therefore, attention should be paid to control the factors favoring the microbial growth in order to safeguard the health of exposed inhabitants.

Gallic acid protects against the COPD-linked lung inflammation and emphysema in mice.

Gallic acid (GA) a naturally occurring phenolic compound, known to possess antioxidant/anti-inflammatory activities. The aim of the present work was to investigate the beneficial effects of GA against COPD-linkedlung inflammation/emphysema by utilizing elastase (ET) and cigarette smoke (CS)-induced mice model. Male BALB/c mice were treated with ET (1U/mouse) or exposed to CS (9 cigarettes/day for 4days). GA administration was started 7days (daily) prior to ET/CS exposure. Broncho-alveolar lavage was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung homogenate was assessed for MPO activity/GSH/MDA/protein carbonyls. Further, Lung tissue was subjected to semi-quantitative RT-PCR, immunoblotting, and histological analysis. GA suppressed the ET-induced neutrophil infiltration, elevated MPO activity and production of pro-inflammatory cytokines (IL-6/TNF-α/IL-1β) at 24h. Reduced inflammation was accompanied with normalization of redox balance as reflected by ROS/GSH/MDA/protein carbonyl levels. Further, GA suppressed phosphorylation of p65NF-κB and IκBα along with down-regulation of IL-1β/TNF-α/KC/MIP-2/GCSF genes. Furthermore, GA offered protection against ET-induced airspace enlargement and ameliorated MMP-2/MMP-9. Finally, GA suppressed the CS-induced influx of neutrophils and macrophages and blunted gene expression of TNF-α/MIP-2/KC. Overall, our data show that GA effectively modulates pulmonary inflammation and emphysema associated with COPD pathogenesis in mice.

Diabetes mellitus type 2 reduces the viability, proliferation, and angiogenic marker of adipose-derived stem cells cultured in low-glucose anti-oxidant-serum supplemented medium

Introduction: Hyperglycemia in diabetic patients induces elevated pro-inflammatory cytokine production, resulting in cellular damage, which may affect the regenerative function of mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs). Identifying the effect of diabetes on ADSCs and optimization of culture conditions is therefore an important starting point for the application of autologous stem cells to improve clinicial outcomes. The aim of this study was to investigate the effect of diabetes on ADSCs that cultured in low-glucose anti-oxidant-serum supplemented medium.&#x0D; Methods: In this study, freshly isolated stromal vascular fraction (SVF) and expanded ADSCs were compared between diabetic and non-diabetic donors. SVF were isolated from the abdominal fat, and total viable cells and viability were estimated. Fresh SVF were cultured in low-glucose (100 mg/dL) culture medium supplemented with an anti-oxidant and fetal bovine serum (complete culture medium) at a low density for 14 days for the colony formation unit-fibroblast (CFU-F) assay. The remaining SVF were expanded to obtain ADSCs in the complete culture medium, which were evaluated based on MSCs surface marker expression and three lineage differentiation potential. Diabetic and non-diabetic ADSCs were compared with respect to population doubling time and viability after serial passage.&#x0D; Results: Total viable counts (0.97 +/- 0.39 x 109 cells/10 mL of adipose tissue, 0.56 +/- 0.39 x 109 cells/10 mL of adipose tissue, p=0.02, independent t-test), but not viability (98.63 +/- 1.12%, 98.20 +/- 1.21%, p= 0.38, independent t-test), were significantly higher for SVF cells from adipose tissues of non-diabetic donors than diabetic donors. Fewer CFU-F were obtained from cultured diabetic SVF than from non-diabetic SVF. Diabetic and non-diabetic ADSCs had similar differentiation potency and CD73 (99.44 +/- 0.34%, 97.15 +/- 5.37%, p= 0.21, Mann-Whitney U test) and CD90 (97.30 +/- 2.86%, 95.06 +/- 6.32%, p= 0.90, Mann- Whitney U test) expression, but significantly fewer diabetic ADSCs expressed CD105 or endoglin, a marker for angiogenesis (89.91 +/- 7.14%, 57.90 +/- 21.36% for non-diabetic and diabetic groups, p&lt; 0.001, Mann-Whitney U test). Diabetic ADSCs tended to exhibit slower proliferation (4.43 +/- 2.70 days, 3.04 +/- 0.55 days, p= 0.27 in passage 2 (P2); 3.95 +/- 1.55 days, 2.96 +/- 0.91 days, p= 0.21 in P3, independent t-test) and lower viability than those of non-diabetic ADSCs (77.65 +/- 10.61%, 87.13 +/- 10.06%, p= 0.25 in P2; 82.70 +/- 8.07%, 91.15 +/- 3.77%, p= 0.04 in P3, independent t-test). Culture in low-glucose anti-oxidant-serum supplemented medium did not improve CD105 expression (65.14 +/- 5.86%, 71.06 +/- 10.27%, 64.05 +/- 10.04%, p= 0.70, for P1, P2, and P3, respectively, repeated measure ANOVA) and cell proliferation (p= 0.50 for P2 vs. P3, paired t-test) of diabetic ADSCs.&#x0D; Conclusions: Overall, diabetes reduced CD105 expression and ADSCs proliferation, suggesting that the angiogenic potency of diabetic ADSCs is reduced. The diabetic ADSCs in this study were also more prone to cell death caused by handling technique compared to non-diabetic ADSCs. Therefore, more advanced culture techniques should be applied to expand ADSCs from diabetic patients to achieve expected clinical outcomes.&#x0D;

Abstract 61: Obesity-induced T cell senescence contributes to prostate cancer progression

Abstract Obese men are at greater risk of developing an aggressive form of prostate cancer. The T cell population from obese subjects have shown signatures of cell senescence including shortened telomeres and elevated production of proinflammatory cytokines. The objective of our studies is to better understand the cellular and molecular basis for altered T cell anti-prostate tumor immunity in obesity, and whether this altered immune function contributes to the worse outcomes observed in obese patients. To investigate whether obesity induces a pro-inflammatory senescent phenotype in CD4+ T cells, splenic CD4+ T-cells from 10-week-old male C57Bl/6 mice were isolated and, either pre-stimulated or co-stimulated with anti CD3/CD28 and conditioned media (CM) from macrophages stimulated under obese or normo-weight conditions. After 24 hours conditioned media from these CD4+ T cells was collected, and their relative cytokine expression profile was analyzed. The CD28 receptor is expressed on CD4+ T cells and provides co-stimulatory signals required for T cell activation. CD28 activation results in phosphorylation of Akt. Lack of Akt phosphorylation in CD4+ T cells after CD3+/CD28+ stimulation indicates CD4+ T cell dysfunction and senescence. Fresh murine splenic CD4+ T cells were isolated and stimulated with anti CD3/CD28 under obese or control conditions. Akt1 phosphorylation levels were assessed using Western blot analysis. To determine if obese conditions modify the impact of CD4+ T cell dysfunction on prostate cancer cell signal transduction pathways, prostate cancer epithelial cells were exposed to obese or control CD4+ T-cells CM for 30 minutes and western blot analysis was used to evaluate the modulation of key pro- and anti-tumorigenic pathways. Matrigel Invasion chambers were used to determine if obesity-modified CD4+ T cell CM impacts the invasive capacity of prostate epithelial cells. Obese conditions suppressed the expression of IFN-γ and IL-2 in CD4+ T-cells. Data also suggests that T cells exposed to obese conditions suppress STAT1 activation in prostate cancer cells. Additionally, obese conditions up-regulated the expression of IL-6 and IL-10 in CD4+ T-cells when compared to control. Importantly our data also suggests that T cells exposed to obese conditions induce STAT3 activation in prostate cancer cells. Preliminary data also suggests that obesity-modified CD4+ T cell CM impacts the invasive capacity of prostate cancer cells. Obesity may induce CD4+ T cell dysfunction, limiting their tumor suppressing activity. Findings form this study could substantially advance fundamental understanding of the ways in which obesity alters T cells, thereby promoting uncontrolled tumor growth and mortality from prostate cancer. This study could also provide a framework for future translational research aimed at improving the effectiveness of therapies in obese patients. Citation Format: Alejandra De Angulo, Peyton Travis, Christopher Jolly, Linda deGraffenried. Obesity-induced T cell senescence contributes to prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 61.