Murine Ribonuclease 6 Limits Bacterial Dissemination during Experimental Urinary Tract Infection.

INTRODUCTION: The ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTIs). In this study, we generated Rnase6-deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. METHODS: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. RESULTS: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6-deficient macrophages. CONCLUSION: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection.

Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.

Repurposing HDAC inhibitors to enhance ribonuclease 4 and 7 expression and reduce urinary tract infection.

With the emergence of antibiotic-resistant bacteria, innovative approaches are needed for the treatment of urinary tract infections. Boosting antimicrobial peptide expression may provide an alternative to antibiotics. Here, we developed reporter cell lines and performed a high-throughput screen of clinically used drugs to identify compounds that boost ribonuclease 4 and 7 expression (RNase 4 and 7), peptides that have antimicrobial activity against antibiotic-resistant uropathogens. This screen identified histone deacetylase (HDAC) inhibitors as effective RNase 4 and RNase 7 inducers. Validation studies in primary human kidney and bladder cells confirmed pan-HDAC inhibitors as well as the HDAC class I inhibitor, MS-275, induce RNase 4 and RNase 7 to protect human kidney and bladder cells from uropathogenic Escherichia coli. When we administered MS-275 to mice, RNase 4 and 7 expression increased and mice were protected from acute transurethral E. coli challenge. In support of this mechanism, MS-275 treatment increased acetylated histone H3 binding to the RNASE4 and RNASE7 promoters. Overexpression and knockdown of HDAC class I proteins identified HDAC3 as a primary regulator of RNase 4 and 7. These results demonstrate the protective effects of enhancing RNase 4 and RNase 7, opening the door to repurposing medications as antibiotic conserving therapeutics for urinary tract infection.

Chloroquine Induces ROS-mediated Macrophage Migration Inhibitory Factor Secretion and Epithelial to Mesenchymal Transition in ER-positive Breast Cancer Cell Lines.

Breast cancer (BC) is the leading cause of cancer-related death in women in the world. Since tumor cells employ autophagy as a survival pathway, it has been proposed that autophagy inhibition could be beneficial for cancer treatment. There are several onging clinical trials where autophagy is being inhibited (using chloroquine, CQ or hydroxychloroquine, HCQ) along with chemotherapy with promising results. However, there is also in vitro evidence in which autophagy inhibition can induce epithelial to mesenchymal transition (EMT) in cancer cells, indicating that, at least in some cases, this strategy could be detrimental for cancer patients. In this study, we found that the genetic inhibition of autophagy primed cells for EMT by inducing a decrease in E-cadherin protein levels, while CQ treatment decreased E-cadherin levels, induced morphological changes related to EMT, increased EMT-related transcription factor (EMT-TF) expression and migration in estrogen receptor positive (ER +) BC cell lines. Importantly, CQ treatment increased intracellular reactive oxygen species (ROS) which induced the secretion of macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine related to malignancy. Both ROS production and MIF secretion were responsible for the mesenchymal morphology and increased migratory capacity induced by CQ. Our results indicate that CQ treatment increased malignancy by inducing ROS production, MIF secretion and EMT and suggest that autophagy inhibition in ER + BC patients might have detrimental effects. Our data indicates that a careful selection of patients should be performed in order to determine who will benefit the most from autophagy inhibition with available pharmacological agents for the treatment of breast cancer.

Autophagy inhibition in breast cancer cells induces ROS-mediated MIF expression and M1 macrophage polarization.

Autophagy can function as a survival mechanism for cancer cells and therefore, its inhibition is currently being explored as a therapy for different cancer types. For breast cancer, triple negative breast cancer (TNBC) is the subtype most sensitive to the inhibition of autophagy; but its inhibition has also been shown to promote ROS-dependent secretion of macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine. In this work, we explore the role of MIF in breast cancer, the mechanism by which autophagy inhibition promotes MIF secretion and its effects on neighboring cancer cell signaling and macrophage polarization. We analyzed MIF mRNA expression levels in tumors from breast cancer patients from different subtypes and found that Luminal B, HER2 and Basal subtypes, which are associated to high proliferation, displayed high MIF levels. However, MIF expression had no prognostic relevance in any breast cancer subtype. In addition, we found that autophagy inhibition in 66cl4 TNBC cells increased intracellular Reactive Oxygen Species (ROS) levels, which increased MIF expression and secretion. MIF secreted from 66cl4 TNBC cells induced the activation of MIF-regulated pathways in syngeneic cell lines, increasing Akt phosphorylation in 4T1 cells and ERK phosphorylation in 67NR cells. Regarding MIF/ chemokine receptors, higher levels of CD74 and CXCR2 were found in TNBC tumor cell lines when compared to non-tumorigenic cells and CXCR7 was elevated in the highly metastatic 4T1 cell line. Finally, secreted MIF from autophagy deficient 66cl4 cells induced macrophage polarization towards the M1 subtype. Together, our results indicate an important role for the inhibition of autophagy in the regulation of ROS-mediated MIF gene expression and secretion, with paracrine effects on cancer cell signaling and pro-inflammatory repercussions in macrophage M1 polarization. This data should be considered when considering the inhibition of autophagy as a therapy for different types of cancer.

Autophagy Inhibition Induces the Secretion of Macrophage Migration Inhibitory Factor (MIF) with Autocrine and Paracrine Effects on the Promotion of Malignancy in Breast Cancer.

Breast cancer is the main cause of cancer-related death in women in the world. Because autophagy is a known survival pathway for cancer cells, its inhibition is currently being explored in clinical trials for treating several types of malignancies. In breast cancer, autophagy has been shown to be necessary for the survival of cancer cells from the triple negative subtype (TNBC), which has the worst prognosis among breast cancers and currently has limited therapeutic options. Autophagy has also been involved in the regulation of protein secretion and, of importance for this work, the inhibition of autophagy is known to promote the secretion of proinflammatory cytokines from distinct cell types. We found that the inhibition of autophagy in TNBC cell lines induced the secretion of the macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine involved in breast cancer invasion and immunomodulation. MIF secretion was dependent on an increase in reactive oxygen species (ROS) induced by the inhibition of autophagy. Importantly, MIF secreted from autophagy-deficient cells increased the migration of cells not treated with autophagy inhibitors, indicating that autophagy inhibition in cancer cells promoted malignancy in neighboring cells through the release of secreted factors, and that a combinatorial approach should be evaluated for cancer therapy.

Autophagy and Its Relationship to Epithelial to Mesenchymal Transition: When Autophagy Inhibition for Cancer Therapy Turns Counterproductive.

The manipulation of autophagy for cancer therapy has gained recent interest in clinical settings. Although inhibition of autophagy is currently being used in clinical trials for the treatment of several malignancies, autophagy has been shown to have diverse implications for normal cell homeostasis, cancer cell survival, and signaling to cells in the tumor microenvironment. Among these implications and of relevance for cancer therapy, the autophagic process is known to be involved in the regulation of protein secretion, in tumor cell immunogenicity, and in the regulation of epithelial-to-mesenchymal transition (EMT), a critical step in the process of cancer cell invasion. In this work, we have reviewed recent evidence linking autophagy to the regulation of EMT in cancer and normal epithelial cells, and have discussed important implications for the manipulation of autophagy during cancer therapy.

Autophagy and Its Role in Protein Secretion: Implications for Cancer Therapy.

Autophagy is a protein and organelle degradation pathway important for the maintenance of cytoplasmic homeostasis and for providing nutrients for survival in response to stress conditions. Recently, autophagy has been shown to be important for the secretion of diverse proteins involved in inflammation, intercellular signaling, and cancer progression. The role of autophagy in cancer depends on the stage of tumorigenesis, serving a tumor-suppressor role before transformation and a tumor-survival function once a tumor is established. We review recent evidence demonstrating the complexity of autophagy regulation during cancer, considering the interaction of autophagy with protein secretion pathways. Autophagy manipulation during cancer treatment is likely to affect protein secretion andinter-cellular signaling either to the neighboring cancer cells or to the antitumoral immune response. This will be an important consideration during cancer therapy since several clinical trials are trying to manipulate autophagy in combination with chemotherapy for the treatment of diverse types of cancers.