Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Low dietary fiber intake impairs small intestinal Th17 and intraepithelial T cell development over generations.

Dietary fiber strongly impacts the microbiota. Here, we show that a low-fiber diet changes the small intestinal (SI) microbiota and impairs SI Th17, TCRαß+CD8αß+ and TCRαß+CD8αα+ intraepithelial T cell development. We restore T cell development with dietary fiber supplementation, but this defect becomes persistent over generations with constant low-fiber diets. Offspring of low-fiber diet-fed mice have reduced SI T cells even after receiving a fiber-rich diet due to loss of bacteria important for T cell development. In these mice, only a microbiota transplant from a fiber-rich diet-fed mouse and a fiber-rich diet can restore T cell development. Low-fiber diets reduce segmented filamentous bacteria (SFB) abundance, impairing its vertical transmission. SFB colonization and a fiber-rich diet partially restore T cell development. Finally, we observe that low-fiber diet-induced T cell defects render mice more susceptible to Citrobacter rodentium infection. Together, these results demonstrate the importance of fiber to microbiota vertical transmission and host immune system development.

Methamphetamine Compromises the Adaptive B Cell-Mediated Immunity to Antigenic Challenge in C57BL/6 Mice.

Methamphetamine (METH) is a substance of abuse that causes dysregulation of the innate and adaptive immunity in users. B cells are involved in the humoral component of the adaptive immunity by producing and secreting antibodies (Abs). METH modifies Ab production, although limited information on the impact of this psychostimulant on antigen (Ag)-specific humoral immune responses is available. Since T cell-dependent and T cell-independent Ags are involved in the activation of B lymphocytes, we explored the role of METH on humoral immunity to ovalbumin (OVA; T cell-dependent) and bacterial lipopolysaccharide (LPS; T cell-independent) in C57BL/6 mice. We demonstrated that METH extends the infiltration of B cells into pulmonary and splenic tissues 7 days post-Ag challenge. METH impairs Ab responses in the blood of animals challenged with OVA and LPS. Furthermore, METH diminishes the expression and distribution of IgM on B cell surface, suggesting a possible detrimental impact on users' humoral immunity to infection or autoimmunity.

Methamphetamine alters the TLR4 signaling pathway, NF-κB activation, and pro-inflammatory cytokine production in LPS-challenged NR-9460 microglia-like cells.

Methamphetamine (METH) is a major public health and safety problem worldwide. METH is psychostimulant that activates microglia via the toll-like receptor (TLR) 4/MD2 complex, modulating the abundant production of pro-inflammatory cytokines in the central nervous system (CNS). The TLR4/MD2 complex on the surface of microglia recognizes pathogen-associated molecular patterns such as lipopolysaccharide (LPS) resulting in brain tissue inflammation and neuronal damage. Since METH has been associated with microglia-induced neurotoxicity, we hypothesized that METH impairs the expression of TLR4 and activation of NF-κB in NR-9460 microglia-like cells after LPS challenge. We demonstrated that METH decreases the distribution and expression of TLR4 receptors on the surface of microglia-like cells after incubation with endotoxin. Moreover, METH impairs the TLR4/MD2 complex signaling pathways, compromises the activation of NF-κB, and reduces the production of pro-inflammatory mediators in microglia-like cells upon LPS stimulation. Interestingly, microglia-like cells treated with METH and challenged with LPS showed considerable cellular morphological changes including enlarged nuclei and ruffled surface. Our results suggest that METH may have a significant impact on microglial-induced neuroinflammation, neurotoxicity, and the CNS defense against infection. It also highlights the importance of studying the effects of METH on the molecular and cellular components of users' CNS immunity. Finally, animal studies exploring the role of METH on the effectors functions of microglia after antigenic exposure are necessary to understand drug-related inflammation and neural damage in users.

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/- . These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.