Non-resolving inflammatory monocytes/macrophages are critically involved in the pathogenesis of chronic inflammatory diseases. However, mechanisms of macrophage polarization are not well understood, thus hindering the development of effective strategies to promote inflammation resolution. In this study, we report that macrophages polarized by subclinical super-low dose LPS preferentially expressed pro-inflammatory mediators such as ccl2 (which encodes the protein monocyte chemo attractant protein-1) with reduced expression of anti-inflammatory/homeostatic mediators such as slc40a1 (which encodes the protein ferroportin-1). We observed significantly elevated levels of the autophagy-associated and pro-inflammatory protein p62 in polarized macrophages, closely correlated with the inflammatory activation of ccl2 gene expression. In contrast, we noted a significant increase of ubiquitinated/inactive nuclear-erythroid-related factor 2 (NRF2), consistent with reduced slc40a1 gene expression in polarized macrophages. Addition of the homeostatic restorative agent phenylbutyrate (4-PBA) effectively reduced cellular levels of p62 as well as ccl2 gene induction by super-low dose LPS. On the other hand, application of 4-PBA also blocked the accumulation of ubiquitinated NRF2 and restored anti-inflammatory slc40a1 gene expression in macrophages. Together, our study provides novel insights with regard to macrophage polarization and reveals 4-PBA as a promising molecule in restoring macrophage homeostasis.

Subclinical endotoxemia [low levels of bacterial endotoxin (LPS) in the blood stream] has been correlated with chronic inflammatory diseases, with less-understood mechanisms. We have previously shown that chronic exposure to super low doses of LPS polarizes monocytes/macrophages to a pro-inflammatory state characterized by up-regulation of pro-inflammatory regulators such as p62 and simultaneous down-regulation of anti-inflammatory/resolving regulators such as Nrf2. Building upon this observation, here we show that chronic exposure to super-low doses of LPS leads to accumulation of the Nrf2-inhibitory protein Keap1 in murine monocytes. This is accompanied by increases of p62 and MLKL, consistent with a disruption of autolysosome function in polarized monocytes challenged by super-low dose LPS. Monocytes subjected to persistent super-low dose LPS challenge also accumulate higher levels of IKKβ. As a consequence, SLD-LPS challenge leads to an inflammatory monocyte state represented by higher expression of the inflammatory marker Ly6C as well as lower expression of the anti-inflammatory marker CD200R. Further analysis revealed that Keap1 levels are significantly enriched in the Ly6C hi pro-inflammatory monocyte population. Finally, we show that the TLR4 signaling adaptor TRAM is essential for these effects. Together our study provides novel insight into signaling mechanisms behind low-grade inflammatory monocyte polarization unique to chronic super-low dose LPS exposure.

The field of innate immunity is witnessing a paradigm shift regarding "memory" and "programming" dynamics. Past studies of innate leukocytes characterized them as first responders to danger signals with no memory. However, recent findings suggest that innate leukocytes, such as monocytes and neutrophils, are capable of "memorizing" not only the chemical nature but also the history and dosages of external stimulants. As a consequence, innate leukocytes can be dynamically programmed or reprogrammed into complex inflammatory memory states. Key examples of innate leukocyte memory dynamics include the development of primed and tolerant monocytes when "programmed" with a variety of inflammatory stimulants at varying signal strengths. The development of innate leukocyte memory may have far-reaching translational implications, as programmed innate leukocytes may affect the pathogenesis of both acute and chronic inflammatory diseases. This review intends to critically discuss some of the recent studies that address this emerging concept and its implication in the pathogenesis of inflammatory diseases.

Inflammation is a host response to infection or damage and is vital for clearing pathogens and host debris. When this resolution fails to occur, chronic inflammation ensues. Chronic inflammation is typically characterized as a low-grade, persistent inflammatory process that can last for months or even years. This differs from acute inflammation, which is typically a fast, robust response to a stimulus followed by resolution with return to homeostasis. Inflammation resolution occurs through a variety of cellular processes and signaling components that act as “brakes” to keep inflammation in check. In cases of chronic inflammation, these “brakes” are often dysfunctional. Due to its prevalent association with chronic diseases, there is growing interest in characterizing these negative regulators and their cellular effects in innate leukocytes. In this review, we aim to describe key cellular and molecular homeostatic regulators of innate leukocytes, with particular attention to the emerging regulatory processes of autophagy and lysosomal fusion during inflammation resolution.

Chronic inflammation is an underlying feature of a variety of highly prevalent diseases such as diabetes, atherosclerosis, inflammatory bowel disease, and cancer. Subclinical endotoxemia has emerged as a common factor correlated with these diseases, potentially contributing to the establishment of chronic inflammation through skewing non-resolving inflammatory monocytes. A better understanding of the underlying mechanisms for the programming of non-resolving inflammatory monocytes may facilitate effective strategies to treat diverse inflammatory diseases. We have observed that subclinical low dose LPS may enable non-resolving inflammatory monocytes through disrupting lysosome fusion and causing accumulation of inflammatory mediators such as p62 and pellino1. Administration of autophagy enhancers such as LiCl can attenuate the effects of low dose LPS, by reducing the levels of pellino1. Together, our data suggest an inhibition of autophagy in response to chronic exposure to subclinical endotoxin. Autophagy is a widely recognized homeostatic cellular process with anti-inflammatory capacity. Further elucidation of the mechanism behind this inhibition may shed light on the link between chronic inflammation and chronic subclinical endotoxin exposure, and provide new avenues for disease treatment.