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OBJECTIVE Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts. METHODS Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates. RESULTS The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor β under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05). CONCLUSION These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.

Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2.

Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in Systemic sclerosis (SSc), and we have shown that macrophages constitute the dominant inflammatory signature in SSc tissue. However, little is known about how these cells contribute to fibrotic activation in SSc. Using a bioinformatics approach, we show that the gene expression profile of blood-derived human SSc macrophages is significantly enriched in SSc patient skin. We characterize the immunophenotype of human SSc macrophages as pro-fibrotic, demonstrating that these cells are activated under basal conditions, expressing elevated levels of surface markers associated with activation and releasing CCL2, IL-6, and TGF-beta. We also show that STAT3 is phosphorylated in macrophage from SSc patient compared to the healthy donor, and the phosphorylation is attenuated after IL-6 blockade, suggesting that increased expression of IL-6 by SSc macrophages accounts, at least in part, for increased signaling. Moreover, our results suggest that activation of SSc macrophages arises from soluble factors, as differentiation of healthy donor monocytes in SSc patient-derived plasma confers the immunophenotype of SSc patient macrophages. For the first time, we show that co-culture of human SSc macrophages with SSc fibroblasts induces fibroblast activation. Collectively, these studies implicate macrophages as likely drivers of fibrosis in SSc and suggest therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immune suppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from tumor-promoting to tumor-inhibiting in vitro. We show now for the first time that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of tumor CD4+Foxp3+ regulatory T cells. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.

Oncolytic viruses, through cancer cell-selective lysis and accompanying release of danger signals that promote immune activation, have demonstrated antitumor efficacy in monotherapy of metastatic melanoma and have shown promising activity in combination with checkpoint inhibitors. We present here the activity of ONCR-148, a recombinant oncolytic Herpes Simplex Virus (oHSV) designed to be a safe and efficacious therapy for the treatment of solid tumors. Neuron-specific miR attenuation of ICP4 gene inhibits viral replication in neurons while preserving its potent oncolytic activity in tumor cells. The antitumor potency of the virus is enhanced by incorporating in its genome a transgene expressing fibroblast activation protein (FAP)- and CD3- bispecific antibody that recruits and re-directs the cytolytic activity of T cells toward FAP-expressing stromal cells. FAP is a cell-surface serine protease that is almost exclusively found in fibroblasts within tumor stroma. However, systemic administration of a FAP-CD3 bispecific antibody was not tolerated due to the toxicity toward FAP-expressing bone marrow mesenchymal cells (Tran et al., 2013. J.Exp.Med, 210(6), 1125-1135). Thus, the local intra-tumoral delivery of FAP-CD3 is an attractive approach to promote tumor lysis and T cell activation against cancerous lesions. To test the in vivo activity of ONCR-148, as FAP is poorly expressed in syngeneic mouse tumor models, we developed a bilateral mouse FAP-expressing MC38 syngeneic colon carcinoma model (MC38-FAP), allowing to test local antitumor activity in oHSV-injected tumors and abscopal efficacy mediated by immune cells in the non-injected tumors. Intra-tumoral administration of ONCR-148 in the MC38-FAP model resulted in tumor growth inhibition of 60% (p Citation Format: Agnieszka Denslow, Brian B. Haines, Michael S. Ball, Jacqueline Gursha, Daniel Wambua, Cecilia Kwong, Lingxin Kong, Allison Colthart, Prajna Behera, Peter Grzesik, Jennifer Lee, Terry Farkaly, Caitlin Goshert, Edward M. Kennedy, Lorena Lerner, Christophe Quéva. Development of ONCR-148, a miR-attenuated oncolytic HSV-1 designed to potently activate antitumor T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1452.

We monitored real-time in vivo levels of serotonin release in the digestive system of intact zebrafish embryos during early development (5 days postfertilization, dpf) using differential pulse voltammetry with implanted carbon fiber microelectrodes modified with carbon nanotubes dispersed in nafion. A detection limit of 1 nM, a linear range between 5 and 200 nM, and a sensitivity of 83.65 nA x microM(-1) were recorded. The microelectrodes were implanted at various locations in the intestine of zebrafish embryos. Serotonin levels of up to 29.9 (+/-1.13) nM were measured in vivo in normal physiological conditions. Measurements were performed in intact live embryos without additional perturbation beyond electrode insertion. The sensor was able to quantify pharmacological alterations in serotonin release and provide the longitudinal distribution of this neurotransmitter along the intestine with high spatial resolution. In the presence of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), concentrations of 54.1 (+/-1.05) nM were recorded while in the presence of p-chloro-phenylalanine (PCPA), a tryptophan hydroxylase inhibitor, the serotonin levels decreased to 7.2 (+/-0.45) nM. The variation of serotonin levels was correlated with immunohistochemical analysis. We have demonstrated the first use of electrochemical microsensors for in vivo monitoring of intestinal serotonin levels in intact zebrafish embryos.

Neurotransmitters (NTs) are substances in the brain which are responsible for the transmission of neurological impulses. Changes in their concentrations are associated with numerous behavioral and physiological processes and neurological disorders. As opposed to the traditional chromatographic and capillary electrophoresis, using electrochemical sensors is a fast and inexpensive way to determine concentrations of NTs. In this study we measure the combination of dopamine (DA) and serotonin (SE) with glassy carbon electrodes and differential pulse voltammetry. The major challenge using this method is to differentiate between different NTs, since the signal obtained from the electrode represents the interactive effect of both NTs present. We address this problem through methods of pattern recognition which relate the voltammetric measurements provided by the sensor to the concentration of individual NTs. Two methods of pattern recognition were applied (PCR and PLS-regression). The best rates of correct classification for the validation sets ranged at 42-62% (DA) and 33-50% (SE). When the ranges for correct prediction were extended to include one level above and below the true concentration level, the rates values ranged at 81-91% (DA) and 91-100%(SE). These findings suggest that pattern recognition can be used to model the interaction between different neurotransmitters to predict actual concentrations of neurotransmitters using voltammetry.