PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

A Novel CD206 Targeting Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice.

Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.

Immune Profile of Exosomes in African American Breast Cancer Patients Is Mediated by Kaiso/THBS1/CD47 Signaling.

African American (AA) women with breast cancer are more likely to have higher inflammation and a stronger overall immune response, which correlate with poorer outcomes. In this report, we applied the nanostring immune panel to identify differences in inflammatory and immune gene expression by race. We observed a higher expression of multiple cytokines in AA patients compared to EA patients, with high expression of CD47, TGFB1, and NFKB1 associated with the transcriptional repressor Kaiso. To investigate the mechanism associated with this expression pattern, we observed that Kaiso depletion results in decreased expression of CD47, and its ligand SIRPA. Furthermore, Kaiso appears to directly bind to the methylated sequences of the THBS1 promotor and repress gene expression. Similarly, Kaiso depletion attenuated tumor formation in athymic nude mice, and these Kaiso-depleted xenograft tissues showed significantly higher phagocytosis and increased infiltration of M1 macrophages. In vitro validation using MCF7 and THP1 macrophages treated with Kaiso-depleted exosomes showed a reduced expression of immune-related markers (CD47 and SIRPA) and macrophage polarization towards the M1 phenotype compared to MCF7 cells treated with exosomes isolated from high-Kaiso cells. Lastly, analysis of TCGA breast cancer patient data demonstrates that this gene signature is most prominent in the basal-like subtype, which is more frequently observed in AA breast cancer patients.

PI3Kγ stimulates a high molecular weight form of myosin light chain kinase to promote myeloid cell adhesion and tumor inflammation.

Myeloid cells play key roles in cancer immune suppression and tumor progression. In response to tumor derived factors, circulating monocytes and granulocytes extravasate into the tumor parenchyma where they stimulate angiogenesis, immune suppression and tumor progression. Chemokines, cytokines and interleukins stimulate PI3Kγ-mediated Rap1 activation, leading to conformational changes in integrin α4ß1 that promote myeloid cell extravasation and tumor inflammation Here we show that PI3Kγ activates a high molecular weight form of myosin light chain kinase, MLCK210, that promotes myosin-dependent Rap1 GTP loading, leading to integrin α4ß1 activation. Genetic or pharmacological inhibition of MLCK210 suppresses integrin α4ß1 activation, as well as tumor inflammation and progression. These results demonstrate a critical role for myeloid cell MLCK210 in tumor inflammation and serve as basis for the development of alternative approaches to develop immune oncology therapeutics.

A Novel CD206 Targeting Peptide Inhibits Bleomycin Induced Pulmonary Fibrosis in Mice.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.

Transcriptional repressor Kaiso promotes epithelial to mesenchymal transition and metastasis in prostate cancer through direct regulation of miR-200c.

The loss of miR-200 family, through DNA methylation, results in cancer cells undergoing an epithelial to mesenchymal transition (EMT), and metastasis. In this study, we established that the transcriptional repressor Kaiso directly binds methylated regions of the miR-200 family, and this is reversed with 5-aza treatment. sh-Kaiso PC-3 cells display increased miR-200-a/b/c, miR-141, and miR-429 expression, with miR-200c demonstrating the most significant increase. Interestingly, overexpression of EGFR or treatment with EGF decreases miR-200c expression and this is reversed after treatment with EGFR specific kinase inhibitor PD153035. However, EGF did not have a significant effect on miR-200c in sh-Kaiso DU-145 or PC-3 cell lines, suggesting Kaiso silences miR-200c through the activation of EGFR signaling. Overexpression of Kaiso in LNCaP cells results in decreased expression of miR-200-a/b/c, miR-141, and miR-429, along with increased expression of ZEB1, p-EGFR and total EGFR levels. Overexpression of miR200c in PC-3 cells results in decreased expression of EGFR, ZEB1, ERK1/2 and Kaiso. Additionally, sh-Kaiso PC-3 demonstrates reduced in vivo tumor formation and metastasis. Thus, our data suggests that EGFR signaling regulates the silencing of miR-200 family through Kaiso binding to methylated regions in the promoter.

Association of Epstein - Barr virus and breast cancer in Eritrea.

BACKGROUND: The oncogenic potential of Epstein-Barr virus (EBV) in breast cancer is being increasingly recognized. Despite some controversies regarding such role, new evidence is suggesting a culpability of EBV in breast cancer, particularly in Africa where the virus has been originally associated with causation of several solid and hematological malignancies. One example is a report from Sudan implicating EBV as a prime etiologic agent for an aggressive type of breast cancer, where nearly 100% of tumor tissues were shown to carry viral signatures. To get a broader view on such association, other nearby countries should be investigated. The present study aims to determine the prevalence and possible associations of the virus in Eritrean breast cancer patients. METHODS: Detection of EBV genome using primers that target Epstein Barr Encoded RNA (EBER) gene and Latent Membrane Protein-1 (LMP-1) gene sequences was performed by polymerase chain reaction (PCR) on DNA samples extracted from 144 formalin fixed paraffin embedded breast cancer tissues and 63 non-cancerous breast tissue as control group. A subset of PCR positive samples was evaluated for EBER gene expression by in situ hybridization (ISH). Expression of Latent Membrane Protein-2a (LMP2a) was also assessed by immunohistochemistry in a subset of 45 samples. RESULTS: Based on PCR results, EBV genome signals were detected in a total of 40 samples (27.77%) as compared to controls (p-value = 0. 0031) with a higher sensitivity when using the EBER primers. Five out of the 14 samples stained by EBER-ISH 35.71% were positive for the virus indicating the presence of the viral genome within the tumor cells. Of those stained for IHC 7 (15.55%) were positive for LMP2a showing low viral protein frequency. CONCLUSIONS: Based on these findings it can be concluded that EBV in Eritrea is associated with a smaller subset of tumors, unlike neighboring Sudan, thus pointing to possible differences in population predisposition and diseases epidemiology.