Toll‐like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report (ATVB 11:1944, 2009) suggested that SFA‐induced TLR activation in cell culture systems is due to contaminants in BSA used for conjugating fatty acids. This report casted doubt about proinflammatory effects of SFAs. Our studies herein demonstrate that sodium palmitate (C16:0) or laurate (C12:0) without BSA conjugation induced phosphorylation of IκBα, JNK, ERK, and NFκB p65 and TLR target gene expression in THP1 monocytes or RAW264.7 macrophages, respectively when cultured in low FBS (0.25%) medium. C12:0 induced NFκB activation through TLR2 dimerized with TLR1 or TLR6, and through TLR4. Since BSA was not used in these experiments, contaminants in BSA have no relevance. Unlike suspension cells (THP‐1), BSA conjugation is required for C16:0 to induce TLR target gene expression in adherent cells (RAW264.7). BSA‐conjugated C16:0 transactivated TLR2 dimerized with TLR1 or TLR6, and through TLR4 as seen with C12:0. These results and additional studies with LPS sequester polymixin B and MyD88−/− macrophages indicated that SFA‐induced activation of TLR2 or TLR4 is a fatty acid‐specific effect, but not due to contaminants in BSA or fatty acid preparations. (USDA‐ARS‐WHNRC Program Funds and NIHDK 064007)

Many studies have shown that TLR4 and TLR2 deficient mice are protected from high fat diet-induced inflammation and insulin resistance, suggesting that saturated fatty acids derived from the high fat diet activate TLR-mediated proinflammatory signaling pathways and induce insulin resistance. However, evidence that palmitic acid, the major dietary saturated fatty acid, can directly activate TLR has not been demonstrated. Here we present multiple lines of evidence showing that palmitic acid directly activates TLR2, a major TLR expressed on human monocytes, by inducing heterodimerization with TLR1 in a NADPH oxidase-dependent manner. Dimerization of TLR2 with TLR1 was inhibited by the n-3 fatty acid docosahexaenoic acid. Activation of TLR2 by palmitic acid leads to expression of pro-IL-1β that is cleaved by caspase-1, which is constitutively present in monocytes, to release mature IL-1β. Our results reveal mechanistic insight about how palmitic acid activates TLR2, upregulates NALP3 expression, and induces inflammasome-mediated-IL-1β production in human monocytes which can trigger enhanced inflammation in peripheral tissues, and suggest that these processes are dynamically modulated by the types of dietary fat we consume.

Lipoxygenases (LOXs) catalyze the stereo-specific peroxidation of polyunsaturated fatty acids (PUFAs) to their corresponding hydroperoxy derivatives. Human macrophages express two arachidonic acid (AA) 15-lipoxygenating enzymes classified as ALOX15 and ALOX15B. ALOX15, which was first described in 1975, has been extensively characterized and its biological functions have been investigated in a number of cellular systems and animal models. In macrophages, ALOX15 functions to generate specific phospholipid (PL) oxidation products crucial for orchestrating the nonimmunogenic removal of apoptotic cells (ACs) as well as synthesizing precursor lipids required for production of specialized pro-resolving mediators (SPMs) that facilitate inflammation resolution. The discovery of ALOX15B in 1997 was followed by comprehensive analyses of its structural properties and reaction specificities with PUFA substrates. Although its enzymatic properties are well described, the biological functions of ALOX15B are not fully understood. In contrast to ALOX15 whose expression in human monocyte-derived macrophages is strictly dependent on Th2 cytokines IL-4 and IL-13, ALOX15B is constitutively expressed. This review aims to summarize the current knowledge on the regulation and functions of ALOX15 and ALOX15B in human macrophages.

Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O 2 ) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1␤. Although palmitate-induced endoplasmic reticulum stress and nuclear factor B pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1␣ and HIF-2␣ alone or in combination failed to reduce IL-6 and only modestly reduced IL-1␤ gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1␤ and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.

Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann–Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRβ in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs.

Macrophages in the tumor microenvironment respond to complex cytokine signals. How these responses shape the phenotype of tumor-associated macrophages (TAMs) is incompletely understood. Here we explored how cytokines of the tumor milieu, interleukin (IL)-6 and IL-4, interact to influence target gene expression in primary human monocyte-derived macrophages (hMDMs). We show that dual stimulation with IL-4 and IL-6 synergistically modified gene expression. Among the synergistically induced genes are several targets with known pro-tumorigenic properties, such as CC-chemokine ligand 18 (CCL18), transforming growth factor alpha (TGFA) or CD274 (programmed cell death 1 ligand 1 (PD-L1)). We found that transcription factors of the signal transducer and activator of transcription (STAT) family, STAT3 and STAT6 bind regulatory regions of synergistically induced genes in close vicinity. STAT3 and STAT6 co-binding further induces the basic leucine zipper ATF-like transcription factor (BATF), which participates in synergistic induction of target gene expression. Functional analyses revealed increased MCF-7 and MDA-MB 231 tumor cell motility in response to conditioned media from co-treated hMDMs compared to cells incubated with media from single cytokine-treated hMDMs. Flow cytometric analysis of T cell populations upon co-culture with hMDMs polarized by different cytokines indicated that dual stimulation promoted immunosuppressive properties of hMDMs in a PD-L1-dependent manner. Analysis of clinical data revealed increased expression of BATF together with TAM markers in tumor stroma of breast cancer patients as compared to normal breast tissue stroma. Collectively, our findings suggest that IL-4 and IL-6 cooperate to alter the human macrophage transcriptome, endowing hMDMs with protumorigenic properties. ARTICLE HISTORY

Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds.Nuclear DNA has long been considered the primary target for bioreductive anticancer drugs. These agents, which include the mitomycins, anthracyclines, nitroimidazoles, quinones, and nitrogen mustards, are used by oncologists primarily because of their toxicity toward radiation-insensitive hypoxic fractions in solid tumors (1-4). The historical mechanism for bioreductive drugs, based on studies with the prototype mitomycin C (MMC), 3 involves intracellular activation, binding G/C-rich regions of nuclear DNA, and subsequent cell death (5-7). Many aspects of this hypothesis, however remain problematic and unsubstantiated.The initial evidence for an MMC interaction with DNA was presented in the early 1960s and demonstrated cross-linking of purified bacterial DNA in the presence of cell lysates after exposure to MMC in vitro (5,6,8,9). Subsequent studies using labeled MMC or the MMC analogue, porfiromycin, in various cell-free or in situ conditions have described the metabolic activation, chemical intermediates, and drug-DNA adducts formed from these exposures (9 -11). MMC-DNA adducts have also been detected in various studies using cultured cell lines, but these DNA-drug interactions have only been demonstrated following exposure to extremely high concentrations of MMC (12, 13). Other studies using very high concentrations of MMC have also shown MMC-DNA adducts when tested in cultured cell lines transfected with MMC-activating enzymes modified to be targeted to the nucleus (21, 22). Thus, all direct evidence for a MMC-DNA adduct comes fro...

Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.