Tumor-Derived Retinoic Acid Regulates Intratumoral Monocyte Differentiation to Promote Immune Suppression.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor-associated macrophages (TAMs) rather than immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we find that the TME induces tumor cells to produce retinoic acid (RA), which polarizes intratumoral monocyte differentiation toward TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production in tumor cells or pharmacologic inhibition of RA signaling within TME increases stimulatory monocyte-derived cells, enhances T cell-dependent anti-tumor immunity, and synergizes with immune checkpoint blockade. Furthermore, an RA-responsive gene signature in human monocytes correlates with an immunosuppressive TME in multiple human tumors. RA has been considered as an anti-cancer agent, whereas our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

Monoclonal antibody-mediated neutralization of Clostridioides difficile toxin does not diminish induction of the protective innate immune response to infection.

Clostridioides difficile infection causes pathology that ranges in severity from diarrhea to pseudomembranous colitis. Toxin A and Toxin B are the two primary virulence factors secreted by C. difficile that drive disease severity. The toxins damage intestinal epithelial cells leading to a loss of barrier integrity and induction of a proinflammatory host response. Monoclonal antibodies (mAbs) that neutralize Toxin A and Toxin B, actoxumab and bezlotoxumab, respectively, significantly reduce disease severity in a murine model of C. difficile infection. However, the impact of toxin neutralization on the induction and quality of the innate immune response following infection is unknown. The goal of this study was to define the quality of the host innate immune response in the context of anti-toxin mAbs therapy. At day 2 post-infection, C. difficile-infected, mAbs-treated mice had significantly less disease compared to isotype-treated mice despite remaining colonized with C. difficile. C. difficile-infected mAbs-treated mice still exhibited marked neutrophil infiltration and induction of a subset of proinflammatory cytokines within the intestinal lamina propria following infection that is comparable to isotype-treated mice. Furthermore, both mAbs and isotype-treated mice had an increase in IL-22-producing ILCs in the intestine following infection. MAbs-treated mice exhibited increased infiltration of eosinophils in the intestinal lamina propria, which has been previously reported to promote a protective host response following C. difficile infection. These findings show that activation of host protective mechanisms remain intact in the context of monoclonal antibody-mediated toxin neutralization.

Relationship between arsenic skin lesions and the age of natural menopause.

BACKGROUND: Chronic exposure to arsenic is associated with neoplastic, cardiovascular, endocrine, neuro-developmental disorders and can have an adverse effect on women's reproductive health outcomes. This study examined the relationship between arsenic skin lesions (a hallmark sign of chronic arsenic poisoning) and age of natural menopause (final menopausal period) in populations with high levels of arsenic exposure in Bangladesh. METHODS: We compared menopausal age in two groups of women--with and without arsenic skin lesions; and presence of arsenic skin lesions was used as an indicator for chronic arsenic exposure. In a cross-sectional study, a total of 210 participants were randomly identified from two ongoing studies--participants with arsenic skin lesions were identified from an ongoing clinical trial and participants with no arsenic skin lesions were identified from an ongoing cohort study. Mean age of menopause between these two groups were calculated and compared. Multivariable linear regression was used to estimate the relationship between the status of the arsenic skin lesions and age of natural menopause in women. RESULTS: Women with arsenic skin lesions were 1.5 years younger (p <0.001) at the time of menopause compared to those without arsenic skin lesions. After adjusting with contraceptive use, body mass index, urinary arsenic level and family history of premature menopause, the difference between the groups' age at menopause was 2.1 years earlier (p <0.001) for respondents with arsenic skin lesions. CONCLUSIONS: The study showed a statistically significant association between chronic exposure to arsenic and age at menopause. Heavily exposed women experienced menopause two years earlier than those with lower or no exposure.

Clostridioides difficile Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

Clostridioides difficile Infection: Diagnosis and Treatment Challenges.

Clostridioides difficile is the most important cause of healthcare-associated diarrhea in the United States. The high incidence and recurrence rates of C. difficile infection (CDI), associated with high morbidity and mortality, pose a public health challenge. Although antibiotics targeting C. difficile bacteria are the first treatment choice, antibiotics also disrupt the indigenous gut flora and, therefore, create an environment that is favorable for recurrent CDI. The challenge of treating CDI is further exacerbated by the rise of antibiotic-resistant strains of C. difficile, placing it among the top five most urgent antibiotic resistance threats in the USA. The evolution of antibiotic resistance in C. difficile involves the acquisition of new resistance mechanisms, which can be shared among various bacterial species and different C. difficile strains within clinical and community settings. This review provides a summary of commonly used diagnostic tests and antibiotic treatment strategies for CDI. In addition, it discusses antibiotic treatment and its resistance mechanisms. This review aims to enhance our current understanding and pinpoint knowledge gaps in antimicrobial resistance mechanisms in C. difficile, with an emphasis on CDI therapies.

Advances in Development of Novel Therapeutic Strategies against Multi-Drug Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) with multi-drug resistance (MDR) is a major cause of serious healthcare-associated infections, leading to high morbidity and mortality. This opportunistic pathogen is responsible for various infectious diseases, such as those seen in cystic fibrosis, ventilator-associated pneumonia, urinary tract infection, otitis externa, and burn and wound injuries. Due to its relatively large genome, P. aeruginosa has great diversity and can use various molecular mechanisms for antimicrobial resistance. For example, outer membrane permeability can contribute to antimicrobial resistance and is determined by lipopolysaccharide (LPS) and porin proteins. Recent findings on the regulatory interaction between peptidoglycan and LPS synthesis provide additional clues against pathogenic P. aeruginosa. This review focuses on recent advances in antimicrobial agents and inhibitors targeting LPS and porin proteins. In addition, we explore current and emerging treatment strategies for MDR P. aeruginosa, including phages, vaccines, nanoparticles, and their combinatorial therapies. Novel strategies and their corresponding therapeutic agents are urgently needed for combating MDR pathogens.

Host Immune Responses to Clostridioides difficile Infection and Potential Novel Therapeutic Approaches.

Clostridioides difficile infection (CDI) is a leading nosocomial infection, posing a substantial public health challenge within the United States and globally. CDI typically occurs in hospitalized elderly patients who have been administered antibiotics; however, there has been a rise in the occurrence of CDI in the community among young adults who have not been exposed to antibiotics. C. difficile releases toxins, which damage large intestinal epithelium, leading to toxic megacolon, sepsis, and even death. Unfortunately, existing antibiotic therapies do not always prevent these consequences, with up to one-third of treated patients experiencing a recurrence of the infection. Host factors play a crucial role in the pathogenesis of CDI, and accumulating evidence shows that modulation of host immune responses may potentially alter the disease outcome. In this review, we provide an overview of our current knowledge regarding the role of innate and adaptive immune responses on CDI outcomes. Moreover, we present a summary of non-antibiotic microbiome-based therapies that can effectively influence host immune responses, along with immunization strategies that are intended to tackle both the treatment and prevention of CDI.

Immunological consequences of microbiome-based therapeutics.

The complex network of microscopic organisms living on and within humans, collectively referred to as the microbiome, produce wide array of biologically active molecules that shape our health. Disruption of the microbiome is associated with susceptibility to a range of diseases such as cancer, diabetes, allergy, obesity, and infection. A new series of next-generation microbiome-based therapies are being developed to treat these diseases by transplanting bacteria or bacterial-derived byproducts into a diseased individual to reset the recipient's microbiome and restore health. Microbiome transplantation therapy is still in its early stages of being a routine treatment option and, with a few notable exceptions, has had limited success in clinical trials. In this review, we highlight the successes and challenges of implementing these therapies to treat disease with a focus on interactions between the immune system and microbiome-based therapeutics. The immune activation status of the microbiome transplant recipient prior to transplantation has an important role in supporting bacterial engraftment. Following engraftment, microbiome transplant derived signals can modulate immune function to ameliorate disease. As novel microbiome-based therapeutics are developed, consideration of how the transplants will interact with the immune system will be a key factor in determining whether the microbiome-based transplant elicits its intended therapeutic effect.

TGFß and Hippo Pathways Cooperate to Enhance Sarcomagenesis and Metastasis through the Hyaluronan-Mediated Motility Receptor (HMMR).

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFß signaling. Therefore, we investigated crosstalk between YAP1 and TGFß resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFß and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFß-YAP1-RHAMM axis prevents vascular migration of tumor cells to distant sites. IMPLICATIONS: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFß cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

Hemozoin produced by mammals confers heme tolerance.

Free heme is cytotoxic as exemplified by hemolytic diseases and genetic deficiencies in heme recycling and detoxifying pathways. Thus, intracellular accumulation of heme has not been observed in mammalian cells to date. Here we show that mice deficient for the heme transporter SLC48A1 (also known as HRG1) accumulate over ten-fold excess heme in reticuloendothelial macrophage lysosomes that are 10 to 100 times larger than normal. Macrophages tolerate these high concentrations of heme by crystallizing them into hemozoin, which heretofore has only been found in blood-feeding organisms. SLC48A1 deficiency results in impaired erythroid maturation and an inability to systemically respond to iron deficiency. Complete heme tolerance requires a fully-operational heme degradation pathway as haplo insufficiency of HMOX1 combined with SLC48A1 inactivation causes perinatal lethality demonstrating synthetic lethal interactions between heme transport and degradation. Our studies establish the formation of hemozoin by mammals as a previously unsuspected heme tolerance pathway.

Specialized cells, known as red blood cells, are responsible for transporting oxygen to various organs in the body. Each red blood cell contains over a billion molecules of heme which make up the iron containing portion of the hemoglobin protein that binds and transports oxygen. When red blood cells reach the end of their life, they are degraded, and the heme and iron inside them is recycled to produce new red blood cells. Heme, however, is highly toxic to cells, and can cause severe tissue damage if not properly removed. Scavenger cells called macrophages perform this recycling role in the spleen, liver and bone marrow. Collectively, macrophages can process around five million red blood cells every second or about 100 trillion heme molecules. But, it is unclear how they are able to handle such enormous volumes. Macrophages isolated from human and mice have been shown to transport heme from damaged red blood cells using a protein called HRG1. To investigate the role HRG1 plays in heme-iron recycling, Pek et al. used a gene editing tool known an CRISPR/Cas9 to remove the gene for HRG1 from the macrophages of mice. If HRG1 is a major part of this process, removing the gene should result in a build-up of toxic heme and eventual death of the mouse. But, rather than dying of heme-iron overload as expected, these mutant mice managed to survive. Pek et al. found that despite being unable to recycle heme, these mice were still able to make new red blood cells as long as they had a diet that was rich in iron. However, the darkening color of the spleen, bone marrow, and liver in these HRG1 deficient mice indicated that these mice were still accumulating high levels of heme. Further experiments revealed that these mice protected themselves from toxicity by converting the excess heme into crystals called hemozoin. This method of detoxification is commonly seen in blood-feeding parasites, and this is the first time it has been observed in a mammal. These crystals invite new questions about how mammals recycle heme and what happens when this process goes wrong. The next step is to ask whether humans also start to make hemozoin if the gene for HRG1 is faulty. If so, this could open a new avenue of exploration into treatments for red blood cell diseases like anemia and iron overload.

YAP1-Mediated Suppression of USP31 Enhances NFκB Activity to Promote Sarcomagenesis.

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705-20. ©2018 AACR.

The Heme Connection: Linking Erythrocytes and Macrophage Biology.

Erythroid function and development is intimately linked to macrophages. The primary function of erythrocytes is oxygen delivery, which is mediated by iron-containing hemoglobin. The major source of this iron is a recycling pathway where macrophages scavenge old and damaged erythrocytes to release iron contained within the heme moiety. Macrophages also promote erythropoiesis by providing a supportive niche in the bone marrow as an integral component of "erythorblastic islands." Importantly, inflammation leads to alterations in iron handling by macrophages with significant impact on iron homeostasis and erythropoiesis. The importance of macrophages in erythropoiesis and iron homeostasis is well established and has been extensively reviewed. However, this developmental relationship is not one way, and erythrocytes can also regulate macrophage development and function. Erythrocyte-derived heme can induce the development of iron-recycling macrophages from monocytes, engage pattern recognition receptors to activate macrophages, and act as ligand for specific nuclear receptors to modulate macrophage function. Here, we discuss the role of heme as a signaling molecule impacting macrophage homeostasis. We will review these actions of heme within the framework of our current understanding of the role of micro-environmental factors in macrophage development and function.

Synthesis, structure-activity relationship studies, and antibacterial evaluation of 4-chromanones and chalcones, as well as olympicin A and derivatives.

On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 µg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2',4'-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.