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Gap junctional intercellular communication (GJIC) is a homeostatic process mediated by membrane channels composed of a protein family known as connexins. Alterations to channel activity can modulate suppression or facilitation of cancer progression. These varying roles are influenced by the cancer cell genetic profile and the context-dependent mechanisms of a dynamic extracellular environment that encompasses fluctuations to nutrient availability. To better explore the effects of altered cellular metabolism on GJIC in breast cancer, we generated a derivative of the triple-negative breast cancer cell line MDA-MB-231 optimized for growth in low-glucose. Reduced availability of glucose is commonly encountered during tumor development and leads to metabolic reprogramming in cancer cells. MDA-MB-231 low-glucose adapted cells exhibited a larger size with improved cell–cell contact and upregulation of cadherin-11. Additionally, increased protein levels of connexin 43 and greater plasma membrane localization were observed with a corresponding improvement in GJIC activity compared to the parental cell line. Since GJIC has been shown to affect cellular invasion in multiple cancer cell types, we evaluated the invasive qualities of these cells using multiple three-dimensional Matrigel growth models. Results of these experiments demonstrated a significantly more invasive phenotype. Moreover, a decrease in invasion was noted when GJIC was inhibited. Our results indicate a potential response of triple-negative breast cancer cells to reduced glucose availability that results in changes to GJIC and invasiveness. Delineation of this relationship may help elucidate mechanisms by which altered cancer cell metabolism affects GJIC and how cancer cells respond to nutrient availability in this regard. Supplementary material The online version of this article (10.1007/s12079-020-00601-3) contains supplementary material, which is available to authorized users.

ObjectivesSyntenin-1, a novel endogenous ligand, was discovered to be enriched in rheumatoid arthritis (RA) specimens compared with osteoarthritis synovial fluid and normal synovial tissue (ST). However, the cellular origin, immunoregulation and molecular mechanism of syntenin-1 are undescribed in RA.MethodsRA patient myeloid and lymphoid cells, as well as preclinical models, were used to investigate the impact of syntenin-1/syndecan-1 on the inflammatory and metabolic landscape.ResultsSyntenin-1 and syndecan-1 (SDC-1) co-localise on RA ST macrophages (MΦs) and endothelial cells. Intriguingly, blood syntenin-1 and ST SDC-1 transcriptome are linked to cyclic citrullinated peptide, erythrocyte sedimentation rate, ST thickness and bone erosion. Metabolic CD14+CD86+GLUT1+MΦs reprogrammed by syntenin-1 exhibit a wide range of proinflammatory interferon transcription factors, monokines and glycolytic factors, along with reduced oxidative intermediates that are downregulated by blockade of SDC-1, glucose uptake and/or mTOR signalling. Inversely, IL-5R and PDZ1 inhibition are ineffective on RA MΦs-reprogrammed by syntenin-1. In syntenin-1-induced arthritis, F4/80+iNOS+RAPTOR+MΦs represent glycolytic RA MΦs, by amplifying the inflammatory and glycolytic networks. Those networks are abrogated in SDC-1-/-animals, while joint prorepair monokines are unaffected and the oxidative metabolites are moderately replenished. In RA cells and/or preclinical model, syntenin-1-induced arthritogenicity is dependent on mTOR-activated MΦ remodelling and its ability to cross-regulate Th1 cells via IL-12 and IL-18 induction. Moreover, RA and joint myeloid cells exposed to Syntenin-1 are primed to transform into osteoclasts via SDC-1 ligation and RANK, CTSK and NFATc1 transcriptional upregulation.ConclusionThe syntenin-1/SDC-1 pathway plays a critical role in the inflammatory and metabolic landscape of RA through glycolytic MΦ and Th1 cell cross-regulation (graphical abstract).

Recent studies show a connection between glycolysis and inflammatory response in rheumatoid arthritis (RA) macrophages (MΦs) and fibroblasts (FLS). Yet, it is unclear which pathways could be targeted to rebalance RA MΦs and FLS metabolic reprogramming. To identify novel targets that could normalize RA metabolic reprogramming, TLR7-mediated immunometabolism was characterized in RA MΦs, FLS and experimental arthritis. We uncovered that GLUT1, HIF1α, cMYC, LDHA and lactate were responsible for the TLR7-potentiated metabolic rewiring in RA MΦs and FLS, which was negated by IRAK4i. While in RA FLS, HK2 was uniquely expanded by TLR7 and negated by IRAK4i. Conversely, TLR7-driven hypermetabolism, non-oxidative PPP (CARKL) and oxidative phosphorylation (PPARγ) were narrowly dysregulated in TLR7-activated RA MΦs and FLS and was reversed by IRAK4i. Consistently, IRAK4i therapy disrupted arthritis mediated by miR-Let7b/TLR7 along with impairing a broad-range of glycolytic intermediates, GLUT1, HIF1α, cMYC, HK2, PFKFB3, PKM2, PDK1 and RAPTOR. Notably, inhibition of the mutually upregulated glycolytic metabolites, HIF1α and cMYC, was capable of mitigating TLR7-induced inflammatory imprint in RA MΦs and FLS. In keeping with IRAK4i, treatment with HIF1i and cMYCi intercepted TLR7-enhanced IRF5 and IRF7 in RA MΦs, distinct from RA FLS. Interestingly, in RA MΦs and FLS, IRAK4i counteracted TLR7-induced CARKL reduction in line with HIF1i. Whereas, cMYCi in concordance with IRAK4i, overturned oxidative phosphorylation via PPARγ in TLR7-activated RA MΦs and FLS. The blockade of IRAK4 and its interconnected intermediates can rebalance the metabolic malfunction by obstructing glycolytic and inflammatory phenotypes in RA MΦs and FLS.

Rheumatoid arthritis (RA) is a chronic inflammatory disease involving dysfunctional lymphatic circulation leading to edema. The adjuvant-induced arthritis (AIA) rat model was used to study the effect of lymphatic pump treatment (LPT) on the expression levels of inflammatory cytokines and lymphangiogenesis within the draining popliteal lymph nodes (PLN). PLN from both control and LPT treatment groups were harvested, and their cDNA were analyzed by qPCR to determine the levels of cytokines, VEGF-C, and VEGFR-3. Additionally, immunohistochemistry was used to visualize the expression of VEGF-C and VEGFR-3 and flow cytometry was used to quantify leukocyte cell types in the PLN. The LPT group showed a decrease in ankle circumference and arthritis grade a few days after the initiation of treatment. Flow cytometry of popliteal lymph nodes showed an increase in CD8, CD4, and CD3 cells in arthritic rats, however cell populations were not significantly different between treatment groups. The qPCR data indicated that the LPT group had a slight reduction in levels of IL-17a, IL-4, IL-6, and IL-1β. IHC showed that the PLN from the LPT group had the highest expression area of VEGF-C and the lowest expression area of VEGFR-3 compared to control. In conclusion, The LPT group showed a reduction in ankle circumference and arthritis score, suggesting that LPT helped alleviate edema and inflammation. When compared to control, the LPT group had lower expression of pro-inflammatory cytokines and elevated levels of VEGF-C, an important lymphatic vessel growth factor, in PLN. Thus, the data suggests that LPT may elevate the swelling and inflammation in rat AIA through the reduction of inflammatory cytokines and the elevation of a lymphatic growth factor.

Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0–1000 nM), AZD1152-HQPA (0–1000 nM), and AMG 900 (0–1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC50 values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC50 = 3.7 nM) > AZD1152-HQPA (EC50 = 43.4 nM) > MK-5108 (EC50 = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC50 = 6.5 nM) > AZD1152-HQPA (EC50 = 74.5 nM) > MK-5108 (EC50 = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0–1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.

Flares of joint inflammation and resistance to currently available biologic therapeutics in rheumatoid arthritis (RA) patients could reflect activation of innate immune mechanisms. Herein, we show that a TLR7 GU-rich endogenous ligand, miR-Let7b, potentiates synovitis by amplifying RA monocyte and fibroblast (FLS) trafficking. miR-Let7b ligation to TLR7 in macrophages (MΦs) and FLSs expanded the synovial inflammatory response. Moreover, secretion of M1 monokines triggered by miR-Let7b enhanced Th1/Th17 cell differentiation. We showed that IRAK4 inhibitor (i) therapy attenuated RA disease activity by blocking TLR7-induced M1 MΦ or FLS activation, as well as monokine-modulated Th1/Th17 cell polarization. IRAK4i therapy also disrupted RA osteoclastogenesis, which was amplified by miR-Let7b ligation to joint myeloid TLR7. Hence, the effectiveness of IRAK4i was compared with that of a TNF inhibitor (i) or anti-IL-6R treatment in collagen-induced arthritis (CIA) and miR-Let7b-mediated arthritis. We found that TNF or IL-6R blocking therapies mitigated CIA by reducing the infiltration of joint F480(+)iNOS(+) MΦs, the expression of certain monokines, and Th1 cell differentiation. Unexpectedly, these biologic therapies were unable to alleviate miR-Let7b-induced arthritis. The superior efficacy of IRAK4i over anti-TNF or anti-IL-6R therapy in miR-Let7b-induced arthritis or CIA was due to the ability of IRAK4i therapy to restrain the migration of joint F480(+)iNOS(+) MΦs, vimentin(+) fibroblasts, and CD3(+) T cells, in addition to negating the expression of a wide range of monokines, including IL-12, MIP2, and IRF5 and Th1/Th17 lymphokines. In conclusion, IRAK4i therapy may provide a promising strategy for RA therapy by disconnecting critical links between inflammatory joint cells.

Dysregulation of gap junction intercellular communication (GJIC) is a common feature during cancer progression. GJIC is a means of direct cell-cell communication mediated by regulated membrane channels composed of connexin proteins. This communication is frequently lost between primary tumor cells but may be upregulated at secondary metastatic sites with stromal cells. Control of this process by cancer cells has been shown to facilitate aggressive qualities both in vitro and in vivo. During the process of metastasis, cells encounter numerous metabolic challenges that must be overcome, particularly during growth of primary tumors. In this study, we set out to evaluate if changes to cancer cell metabolism affect GJIC in breast cancer cells. To address this question, we generated a metabolic variant of the MDA-MB-231 cell line conditioned to grow in glucose-limiting conditions. These cells were grown in FBS supplemented RPMI with Citation Format: Jennifer C. Jones, Amanda M. Miceli, Mary M. Chaudhry, Mallika A. Jai, Romel N. Pancho, Alan Lazzar, Bradley S. Taylor, Vishnupriya Bodempudi, Prarthana P. Jain, Sheeri Hanjra, Alexander E. Urban, Brian Zanotti, Ellen K. Kohlmeir, Thomas M. Bodenstine. Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5997.

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation mediated by Th-17 cells. Rat adjuvant-induced arthritis (AIA) is a Th-17-dependent RA model with dysfunctional lymphatic circulation. We hypothesized that induction of lymphatic circulation would attenuate arthritic inflammation. We utilized Lymphatic Pump Treatment (LPT), an osteopathic lymphatic manipulation, to determine if inducing lymphatic flow would alleviate arthritic inflammation. LPT was accomplished on AIA rats by gradually increasing compression on the chest wall for 5 seconds followed by a rapid release. This technique was performed on the rats for 1 minute 3X/day over 7 consecutive days following the onset of measurable inflammation on day 14 post adjuvant injection. Ankle caliper measurements, articular index scoring, popliteal lymph nodes, peripheral blood, and spleens were collected. The LPT-treated rats fell into two groups. LPT responders and non-responders. LPT responders had significantly reduced inflammation (as determined by both in ankle circumference and index scoring). These therapeutically treated animals also had significantly decreased IL-17 expression in their draining popliteal lymph nodes. These results along with our previous findings indicate that LPT can reduce arthritis, alter lymphocyte trafficking and differentiation in secondary lymphoid organs during inflammation. These data suggest that induced lymphatic circulation using osteopathic manipulation may serve as a therapy for chronic inflammation in autoimmune disorders and may also be an enhancement to current treatments.

Epstein-Barr virus (EBV) latently infects over 90% of the population and is associated with the development of numerous autoimmune diseases and B cell lymphomas. Therefore, understanding the impact of latency proteins on B cell survival and function is vital. One EBV latency protein, LMP2A, acts as a BCR mimic to promote B cell survival, increase B cell activation, and accelerate tumor development in murine mouse models. Our laboratory identified that LMP2A activates the PI3K/BTK/STAT3 pathway in human B cell lymphomas to increase IL-10 production that promotes survival. However, the ability of LMP2A to activate BTK and STAT3 in non-transformed B cells is unknown. To test this possibility, we returned to the LMP2A transgenic mouse model as a source of resting B cells that express LMP2A. To determine if LMP2A activates STAT3 in the mouse model, resting B cells from wildtype and LMP2A-transgenic mice were isolated and STAT3 phosphorylation was assessed by Western blot analysis. Our data indicate that LMP2A-postitive B cells express higher levels of phosphorylated STAT3 than wildtype controls. We further analyzed whether the LMP2A-dependent activation of STAT3 resulted in an increase in STAT3-dependent targets. Flow cytometric analysis indicates that the addition of STAT3 inhibitors to activated LMP2A-expressing B cells blocked the LMP2A-dependent increase in STAT3 targets in comparison to wildtype controls. These data suggest that LMP2A increases the activation of the oncogene STAT3 in resting B cells, which may contribute to the development of EBV-associated tumors and autoimmune diseases.

Herpes simplex virus type-1 (HSV-1) is a significant pathogen that affects vision by targeting multiple regions in the human eye including iris. Using a focused library of synthetic non-saccharide glycosaminoglycan mimetics (NSGMs), we identified sulfated pentagalloylglucoside (SPGG) as a potent inhibitor of HSV-1 entry and cell-to-cell spread in the primary cultures of human iris stromal (HIS) cells isolated from eye donors. Using in vitro β-galactosidase reporter assay and plaque reduction assay, SPGG was found to inhibit HSV-1 entry in a dosage-dependent manner (IC50 ∼6.0 μM). Interestingly, a pronounced inhibition in HSV-1 entry and spread was observed in HIS cells, or a cell line expressing specific gD-receptor, when virions were pre-treated with mimetics suggesting a possible interaction between SPGG and the HSV-1 glycoprotein. To examine the significance of gD-SPGG interaction, HIS cells were pretreated with SPGG, which showed a significant reduction in gD binding. Taken together, our results provide strong evidence of SPGG being a novel viral entry inhibitor against ocular HSV infection.

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3- O -sulfated heparan sulfate (3- O S HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3- O -sulfotransferase-3 (3- O ST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3- O S HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3- O S HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3- O S HS cells. Taken together, these results highlight the role of 3- O S HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.

We hypothesized that reactive arthritis can be induced in the knees and ankles of wild-type C57BL/6 mice following infection with Chlamydia muridarum through the involvement of inflammatory mediators. Reactive arthritis is a chronic form of arthritis, which can also result in conjunctivitis and inflammation of the genital, urinary, or gastrointestinal systems. C. muridarum is a gram-negative obligate intracellular pathogen which can infect the epithelium of the cervix, urethra, and rectum. The exact pathological mechanism of reactive arthritis is unknown. It is understood to result from CD4+ and CD8+ T cells, and factors including TNF-alpha and perforin. Five groups of C57BL/6J mice were used including a positive control group, CD4+ T cells knockout mice, TNF-alpha knockout mice, perforin knockout mice and TNFRSF knockout mice. Mice from each group were intra-vaginally infected with 5×104 inclusion forming units of C. muridarum, marked as day 0. The mice were swabbed a few days after infection to confirm C. muridarum. The mice from each group were euthanized on day 7 and day 14, and sera was collected and analyzed for cytokine expression using an antibody array. Ankles collected from C57BL/6J positive control mice were histologically processed and graded to determine pathology. C57BL/6J positive control mice showed increased synovial lining thickening 14 days following infection. Cytokine expression from TNF-alpha knockout mice, CD4+ T cells knockout mice, perforin knockout mice, and TNFRSF knockout mice was generally reduced. The results show that chlamydial induced reactive arthritis can be detected in the ankles of C57BL/6J mice and suggest that TNF-alpha and CD4+ T cells may be involved in this inflammatory process.

Ocular herpes simplex virus 1 (HSV-1) infection can lead to multiple complications, including iritis, an inflammation of the iris. Here, we use human iris stroma cells as a novel in vitro model to demonstrate HSV-1 entry and the inflammatory mediators that can damage the iris. The upregulated cytokines observed in this study provide a new understanding of the intrinsic immune mechanisms that can contribute to the onset of iritis.

Rheumatoid arthritis (RA) is characterized by synovial inflammation, cytokine production and lymph node collapse. Lymphatic pump treatment (LPT) is an osteopathic manipulative treatment used to reduce edema through increasing lymphatic circulation. This study examined the therapeutic potential of LPT on rats with adjuvant-induced arthritis (AIA). We hypothesize that LPT would lessen joint inflammation through increasing lymphatic drainage resulting in the reduction of cytokines in synovial space. AIA was induced in female Lewis rats and randomly placed into a LPT groups or sham groups. Experiments were repeated using different LPT protocols varying the frequency and number of LPT treatments. However in each experiment LPT consisted of rhythmically pressing below the rib cage once a second for 30 seconds, while the sham group was held for 30 seconds. Joint inflammation was determined by measuring ankle circumferences and by articular index scoring. Ankles were collected and homogenized to determine changes in synovial cytokine levels by magnetic immunoassays and ELISA. Clustering LPT treatments three-times a day at the peak of joint inflammation had the greatest impact on arthritis. Animals treated with LPT in this manner exhibited significantly reduced ankle edema and inflammation. Additionally, synovial levels of inflammatory cytokines (GRO, IL17, and IL-6) were reduced with LPT. These results indicate that LPT can reduce joint edema and inflammation in a rat model of RA.

Isolated from sponges on the ocean floor, MZA has anti-inflammatory properties which might be beneficial in treating rheumatoid arthritis (RA) or osteoarthritis (OA). Preliminary data suggested that MZA causes RA fibroblast-like synoviocytes (FLS) to detach from gelatin. We hypothesized that MZA decreased the adhesive properties of synovial fibroblasts isolated from RA, OA, or normal joints. All fibroblasts were isolated from tissues collected from patients with IRB approval. Photos of cells were taken at various time points (1, 2, 4, 8, 12, and 24 hrs) and counted for adherence after treatment with various concentrations of MZA or vehicle on gelatin, collagen, or laminin substrates. Flow cytometry was used to determine α1 integrin subunit expression on various cell types, since this subunit is important in binding to adhesive substrates. RA FLS on gelatin appeared most sensitive to MZA, in that 1 µM could significantly detach cells in as little as 8 hrs (n=4, p

Mucin 3 (MUC3) is elevated in rheumatoid arthritis (RA) synovial tissues (STs). We hypothesized that MUC3 is expressed by RA synovial fibroblasts, that it is cytokine inducible and chemotactic for RA synovial fibroblasts. To test this, RA synovial fibroblast MUC3 expression was determined by immunohistochemistry, flow cytometry and Western blot analysis. Levels of MUC3 from arthritic synovial fluids (SFs) were measured by a novel MUC3 ELISA. RA synovial fibroblast chemotaxis to RA SFs was assessed using a modified Boyden chamber assay. RA ST cells stained for both MUC3 and vimentin indicating that RA synovial fibroblasts express MUC3. Flow cytometry experiments showed that most of the RA synovial fibroblast MUC3 is cytoplasmic. Cytokine stimulation of these cells significantly increased MUC3 production (P

MZA is a complex alkaloid compound with anti-inflammatory properties that has recently been synthesized. OA has been characterized to have an inflammatory component involving increased cytokine release by synovial tissue fibroblasts. We hypothesized that MZA treatment of OA fibroblasts could decrease their production of pro-inflammatory cytokines at non-toxic concentrations. OA fibroblasts were isolated with IRB approval, treated with MZA or vehicle, and viability was assessed using multiple methods. OA fibroblast condition media (CM) was collected at various time points after MZA or control treatment, screened by antibody microarrays, and verified by ELISAs. Toxicity studies on OA fibroblasts suggested that concentrations up to 2.5 µM MZA were not toxic using a sensitive flow cytometry kit (results varied in a method-, time-, and concentration-dependent manner). Antibody microarray performed on CM pooled from 5 primary OA fibroblast lines treated with 10 µM MZA for 24 hrs suggested that monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6 were two of the most prominent proteins detected, and MZA appeared to decrease MCP-1 production. ELISAs suggested that concentrations of 0.5 µM MZA and higher significantly decreased production of MCP-1 (n=5; p