miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

It is well known that the nonmalignant stromal components in tumors play pivotal roles in tumor progression and therapeutic responses (1, 2). Macrophages are a major component of tumor microenvironment and display considerable phenotypic plasticity in their effects toward tumor progression (3–5). Classically activated (M1) macrophages often exert direct tumor cytotoxic effects or induce antitumor immune responses by helping present tumor-related antigens (6, 7). In contrast, tumoral cues can polarize macrophages toward alternative activation with immunosuppressive M2 properties (6–8). Numerous studies have firmly established the protumor effects of M2-like tumor-associated macrophages (TAMs) and the association of TAMs with poor prognosis of human cancer (9–11). However, how tumors induce the coordinated molecular and phenotypic changes in TAMs for M2 polarization remains incompletely understood, impeding the designing of TAM-targeting strategies for cancer intervention. In addition, drug delivery also represents a hurdle for therapeutic macrophage reprogramming.Noncoding RNAs, including microRNAs, have been shown to play vital roles in various pathological processes of cancer (12). The microRNA miR-182 has been implicated in various developmental processes and disease conditions (13–15). Particularly, it receives extensive attention in cancer studies. Prevalent chromosomal amplification of miR-182 locus and up-regulation of its expression in tumors have been observed in numerous cancer types including breast cancer, gastric cancer, lung adenocarcinoma, colorectal adenocarcinoma, ovarian carcinoma, and melanoma (16–21). miR-182 expression is also linked to higher risk of metastasis and shorter survival of patients (20, 22–24). Functional studies showed that miR-182 expression in cancer cells plays vital roles in various aspects of cancer malignancy, including tumor proliferation (25–29), migration (30, 31), invasion (16, 32, 33), epithelial-mesenchymal transition (34–36), metastasis (21, 37, 38), stemness (30, 39, 40), and therapy resistance (41, 42). A number of target genes, including FOXO1/3 (18, 21, 43–45), CYLD (46), CADM1 (47), BRCA1 (27, 48), MTSS1 (34), PDK4 (49), and SMAD7 (35), were reported to be suppressed by miR-182 in cancer cells. Our previous work also proved that tumoral miR-182 regulates lipogenesis in lung adenocarcinoma and promotes metastasis of breast cancer (34, 35, 49). Although miR-182 was established as an important regulator of cancer cell malignancy, previous studies were limited, with analyses of miR-182 in cultured cancer cells and transplanted tumors. Thus, the consequences of miR-182 regulation in physiologically relevant tumor models, such as genetically modified mice, have not been shown. More importantly, whether miR-182 also plays a role in tumor microenvironmental cell components is unknown.In this study, we show that miR-182 expression in macrophages can be induced by breast cancer cells and regulates TAM polarization in various tumor models of mice. In addition, miR-182 inhibition with TAM-targeting exosomes demonstrates promising efficacy for cancer treatment.     

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns that can elicit a host defense response through binding to cell-surface Toll-like receptor 4 (TLR4). Systemic inflammatory response syndrome is induced by overstimulation of the innate immune response via LPSs, resulting in severe multiple organ failure, which is a major cause of death worldwide in intensive care units (1). LPS-induced dimerization of TLR4 initiates signal transduction involving the NF-κB– and MyD88-dependent and -independent pathways, thereby contributing to various inflammatory responses (2). Another set of the immune repertoire, which resides in the cytosol and comprises NLRP1, NLRP3, NAIP/NLRC4, and AIM2, is known as the inflammasome. Inflammasomes can be activated in response to a number of well-defined pathogen-derived ligands and physiological aberrations, which in turn trigger caspase-1–mediated pyroptotic death (3, 4). This process has been associated with strengthening the host defense program to eliminate intracellular bacteria.Recently, a cytosolic LPS-sensing pathway involving caspase-4/5 in humans and caspase-11 in mice was termed the noncanonical inflammasome pathway, and this pathway is independent of TLR4 (5–8). LPSs from extracellular bacteria can enter the cytoplasm and trigger caspase-4/5/11–dependent responses. LPSs can be delivered into the cytosol when LPS-containing outer membrane vesicles (OMVs) from gram-negative bacteria are taken up by the cells or when intracellular bacteria escape from the phagosomes that are damaged by host resistant factors such as guanylate-binding protein and HMGB1 or microbe-derived hemolysins (9–12). LPSs comprise three regions: lipid A, core oligosaccharide, and O-polysaccharide (also termed O-antigen). The lipid A moiety binds directly to the caspase-4/5/11 caspase activation and recruitment domain (CARD, also known as prodomain), leading to caspase oligomerization and activation (7). This event likely mimics the proximity-induced dimerization model of initiator caspase activation (13). Furthermore, caspase-4/5/11 executes downstream signaling events via gasdermin D. Activated inflammatory caspase proteolytically cleaves gasdermin D to create an N-terminal fragment that self-oligomerizes and then inserts into the cell membrane to form pores, causing lytic cell death (14–17). Various stimuli have been identified in the caspase-1–mediated canonical-inflammasome signaling pathway (3, 4), but the detailed mechanism underlying noncanonical inflammasome activation mediated by caspase-4/5/11 remains unclear.Galectins, a family of β-galactoside–binding proteins, can decode host-derived complex glycans and are involved in various biological responses (18–23). Galectins are nucleocytoplasmic proteins synthesized without a classical signal sequence, although they can be secreted through unconventional pathways (19, 21, 23, 24). Recent studies have revealed prominent roles of cytosolic galectins in host defense programs (12, 25, 26). The proposed molecular mechanisms involve the binding of galectins to host glycans exposed to the cytosolic milieu upon endosomal or phagosomal membrane damage. In addition to binding host glycans, galectins also recognize microbial glycans, particularly LPSs (27–30). However, the contribution of galectins to the host response through binding to cytosolic LPSs is unknown.Galectin-3 is an ∼30-kDa protein that contains a carbohydrate-recognition domain (CRD) connected to N-terminal proline, glycine, and tyrosine-rich tandem repeats. Upon binding to multivalent glycoconjugates through its CRD, the protein forms oligomers, which is attributable to the self-association property of its N-terminal region (31, 32). Galectin-3 binds to LPSs of various gram-negative bacteria by recognizing their carbohydrate residues (33–36).Although structural information is scarce (37), existing information suggests that ligand-induced oligomerization of caspase CARD is necessary for the activation of inflammatory caspases (7, 38). Therefore, we hypothesized that galectin-3 may be an intracellular LPS sensor that participates in LPS-induced CARD-mediated inflammatory caspase activation. Specifically, highly ordered arrays of LPS–galectin-3 complexes may amplify caspase-4/5/11 oligomerization and activation. Here, we investigated the formation of galectin-3–LPS–caspase-4/11 complexes in cell-based and cell-free systems. Our findings provide evidence regarding a role of galectin-3 as an intracellular mediator in noncanonical inflammasome activation through LPS glycan recognition.     

CD81 costimulation skews CAR transduction toward naive T cells

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Genetic manipulation of T cells has enabled adoptive T cell therapy to be translated to the clinic (1–10). Chimeric antigen receptor (CAR) therapy has evoked recent enthusiasm upon mediating curative outcomes in aggressive, refractory B cell malignancies (1–7, 11–15), leading to Food and Drug Administration approval (16–18). The process of ex vivo transduction and expansion of T cells to express CARs influences the phenotype, function, and ultimate fate of the final CAR T cell product (19–23). Preclinical data in animal models indicate that selecting specific T cell subsets for CAR transduction improves efficacy (21, 22, 24–26). Naive-derived T cells have been shown to exhibit greater replicative capacity, persistence, and antitumor function, compared with both effector- and memory-derived T cells (19, 20, 27). Naive CD4⁺ T cells, specifically, have a critical role in enhancing the cytotoxic effect of the CD8⁺ cooperating central memory cell subset (21). Furthermore, the translational CAR experience demonstrates that the presence of cells consistent with the naive and early memory phenotype in premanufactured T cell products correlates with successful clinical responses in both pediatric and adult B cell leukemia (28–30). Here, we explore if selective activation of naive T cells can result in skewing of transduction toward this specific T cell subset without the need for physical subset sorting.CAR constructs rely on intrinsic costimulatory signals, such as the intracellular domains of CD28 or 41BB, for efficacy (1–9). Here we focus on exogenous costimulatory signals necessary to induce proliferation and permit viral-mediated gene transfer. Prior to CAR transduction and antigen encounter, the majority of T cells are in a state of rest. Resting T cells mandate primary and costimulatory signals for activation (31, 32). CD28 is best known for its ability to costimulate T cells (33–38) and along with CD3 activation renders T cells susceptible to viral transduction (1, 39). CD81 is a member of the tetraspanin family that physically associates with CD4 and CD8 on the surface of T cells. CD81 was shown to have independent costimulatory properties and, when used with anti-CD3 and -CD28 antibodies, preferentially activates naive T cells as compared with effector and memory T cells, despite conserved surface CD81 expression across T cell subsets (40). Tetraspanins have no known cell-surface ligands, and therefore antibodies are used to engage and stimulate them. CD81 is the only tetraspanin whose complete three-dimensional structure has been solved (41). Moreover, the crystal structure of 5A6, the anti-CD81 antibody used in our study, in complex with CD81 has also been most recently solved (42). These authors demonstrate that engagement by this antibody changes the conformation of the large extracellular loop of the CD81 molecule. This conformational change may affect the interaction of CD81 with its associated CD4 and CD8 molecules.Here, we costimulate purified T cells with CD81 as a proof of principle to illustrate that the in vitro activation window prior to CAR transduction can be leveraged to favor transduction of a specific T cell subset.     

Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens

Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.Pattern recognition receptors (PRRs) of the innate immune system are critical for promoting defense against bacterial pathogens (1). Cytosolic PRRs are key for discriminating between pathogenic and nonpathogenic bacteria, because many pathogens access the host cytosol, a compartment where microbial products are typically not found (2). Cytosolic PRRs respond to patterns of pathogenesis that are often associated with virulent bacteria, such as the use of pore-forming toxins or injection of effector molecules through specialized secretion systems (3). A subset of cytosolic PRRs induces the formation of multiprotein complexes known as inflammasomes (4). In mice, the canonical inflammasome activates caspase-1, an inflammatory caspase that mediates cell death and IL-1 family cytokine secretion (5, 6). Additionally, the noncanonical inflammasome activates caspase-11 in response to many gram-negative bacteria (7–14). The canonical and noncanonical inflammasomes differentially regulate release of IL-1α and IL-1β (7). Caspase-11 mediates LPS-induced septic shock in mice (7, 15), and caspase-11 responds to cytoplasmic LPS independent of Toll-like receptor 4 (16, 17).In addition to its pathologic role in septic shock, the noncanonical inflammasome is critical for host defense in mice (11, 18). However, in humans, it is unclear whether an analogous noncanonical inflammasome exists. Whereas mice encode caspase-11, humans encode two putative functional orthologs: caspase-4 and caspase-5 (19–21). All three inflammatory caspases bind directly to LPS in vitro (22). In murine macrophages, caspase-1 and caspase-11 have both distinct and overlapping roles in the release of IL-1α and IL-1β and the induction of cell death (7). However, the precise role of the human inflammatory caspases in the context of infection by bacterial pathogens remains unclear.To elucidate how human inflammasome activation is regulated, we investigated the contribution of inflammatory caspases to the response against gram-negative bacterial pathogens in human macrophages. Here, we show that both canonical caspase-1–dependent and noncanonical caspase-1–independent inflammasomes are activated in primary human macrophages and that caspase-4 mediates caspase-1–independent inflammasome responses against several bacterial pathogens, including Legionella pneumophila, Yersinia pseudotuberculosis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Importantly, noncanonical inflammasome activation in human macrophages is specific for virulent strains of these bacteria that translocate bacterial products into the host cytosol via the virulence-associated type III secretion system (T3SS) or type IV secretion system (T4SS). Thus, caspase-4 is critical for noncanonical inflammasome responses against virulent gram-negative bacteria in human macrophages.     

Anticancer efficacy of monotherapy with antibodies to SIRPα/SIRPβ1 mediated by induction of antitumorigenic macrophages

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPβ1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell–killing activity of macrophages, however. Moreover, knockdown of SIRPβ1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPβ1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPβ1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPβ1 is a potential target for cancer immunotherapy.

Macrophages are innate immune cells that show phenotypic heterogeneity and functional diversity; and they play key roles in development, tissue homeostasis and repair, and in cancer, as well as in defense against pathogens (1–3). In the tumor microenvironment (TME), macrophages are exposed to a variety of stimuli, including cell–cell contact, hypoxia, as well as soluble and insoluble factors such as cytokines, chemokines, metabolites, and extracellular matrix components (2, 4). These environmental cues promote the acquisition by macrophages of protumorigenic phenotypes that facilitate tumor development, progression, and metastasis as well as suppress antitumor immune responses (2, 4). A high density of macrophages within tumor tissue is associated with poor prognosis in patients with various types of cancer, including that of the bladder or breast (5–7). Depletion of macrophages in the TME or the reprogramming of these cells to acquire antitumorigenic phenotypes has been shown to ameliorate the immunosuppressive condition and result in a reduction in tumor burden in both preclinical and clinical studies (2, 4, 8, 9). Macrophages within the TME have therefore attracted much attention as a potential therapeutic target for cancer immunotherapy.Signal regulatory protein α (SIRPα) is a transmembrane protein that possesses one NH₂-terminal immunoglobulin (Ig)-V–like and two Ig-C domains in its extracellular region, as well as immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic region (10, 11). The extracellular region of SIRPα interacts with that of CD47, another member of the Ig superfamily of proteins, with this interaction constituting a means of cell–cell communication. The expression of SIRPα in hematopoietic cells is restricted to the myeloid compartment—including macrophages, neutrophils, and dendritic cells (DCs)—whereas CD47 is expressed in most normal cell types as well as cancer cells (12, 13). The interaction of SIRPα on macrophages with CD47 on antibody (Ab)-opsonized viable cells such as blood cells or cancer cells prevents phagocytosis of the latter cells by the former (13–15), with this negative regulation of macrophages being thought to be mediated by SHP1, a protein tyrosine phosphatase that binds to the cytoplasmic region of SIRPα (14). Indeed, blockade of the CD47–SIRPα interaction by Abs to either SIRPα or CD47, in combination with a tumor-targeting Ab such as rituximab (anti-CD20), was found to enhance the Ab-dependent cellular phagocytosis (ADCP) activity of macrophages for cancer cells that do not express SIRPα, resulting in marked suppression of tumor formation in mice (15–19). Targeting of SIRPα in combination with a tumor-targeting Ab therefore provides a potential approach to cancer immunotherapy dependent on enhancement of the ADCP activity of macrophages for cancer cells. In contrast, the effect of Abs to SIRPα in the absence of a tumor-targeting Ab on the phagocytosis by macrophages of, as well as on tumor formation by, cancer cells that do not express SIRPα was minimal or limited.We have now further examined the antitumor efficacy of a monoclonal Ab (mAb) to mouse SIRPα (MY-1) (20) in immunocompetent mice transplanted subcutaneously with several types of murine cancer cells that do not express SIRPα. This Ab prevents the binding of mouse CD47 to SIRPα and cross-reacts with mouse SIRPβ1 (15). We found that monotherapy with MY-1 efficiently attenuated the growth of tumors formed by bladder or mammary cancer cells. In addition, MY-1 markedly promoted the induction of antitumorigenic macrophages able to target these cancer cells. Furthermore, our results suggest that SIRPβ1 on macrophages likely participated in the antitumorigenic effect of MY-1.     

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography–induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial–macrophage interaction.

Macrophages play an essential role in our innate immune system. They patrol tissues, detect pathogens, and respond to tissue damage (1–3). Macrophages remove pathogens and cellular debris through phagocytosis and encapsulation, and they control downstream processes such as wound healing and tissue regeneration (4, 5). To accommodate the multitude of tasks, macrophages exhibit a high degree of plasticity, which allows them to reversibly adopt different phenotypes within a short period of time (6–8). Macrophage activation is dependent on a variety of bioactive molecules and is usually controlled by complex signaling pathways in vivo. Macrophages are typically classified into the following categories: M0 (resting), M1 (proinflammatory), and M2 (anti-inflammatory). M2 macrophages are further divided into fibrotic (M2a) and regenerative (M2c) subtypes (9). This classification is not definite, as many other activation states exist (10).Adverse immune reactions toward implant materials are known to cause chronic inflammation, tissue loss, aseptic loosening, and fibrotic encapsulation (11, 12). This process is typically associated with significant pain for the patient, the need for repeated surgeries, and implant replacement (13, 14). Recently, materials designed to modulate the immune response have seen intense interest, as they may improve implant durability and integration (15). Macrophages are key cells in this process and are highly sensitive to material surface characteristics such as topography, stiffness, or wettability (16–21). However, how these properties impact macrophage behavior is not fully understood.Macrophage classification is typically performed using techniques such as flow cytometry (FC), analyzing RNA expression, or enzyme-linked immunosorbent assays. These end-point analytical techniques require complex processing and invasive reagents such as antibodies and primers. Cells are not analyzed in their native state, and the focus is on the expression of only predefined marker genes or proteins. As macrophage polarization is in flux, their classification into discrete categories based on a priori defined markers can be problematic (22).Raman microspectroscopy (RM) has emerged as a powerful tool for the investigation of living cells, assessing their physiology and biochemical composition on a single-cell level without the need for processing or staining (23). RM can be used to identify chemical fingerprints or patterns of distinct cellular components such as nucleic acids, proteins, and lipids (24, 25). Previous research has demonstrated that RM can be used to reliably distinguish between different cell phenotypes or stages of the cell cycle based on the spectral information obtained by RM (25–29).In this study, we present an approach for in situ macrophage characterization and monitoring their response to distinct surface features of titanium. By analyzing Raman spectral fingerprints of activated macrophages, we were able to characterize the cell response without detaching or exogenously labeling the cells. Information derived from these data provides valuable insight into macrophage physiology on a level that is not achievable by classical analytical techniques, potentially providing information for the field of macrophage activation. Furthermore, we demonstrate that RM can analyze material-adherent cells and that the resulting spectral information can be used to classify the macrophage response into the existing activation spectrum.     

Eicosanoid regulation of debris-stimulated metastasis

Cancer therapy reduces tumor burden via tumor cell death (“debris”), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E₂ receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death and the most rapidly increasing cancer in the United States (1). Pancreatic cancer is the fourth leading cause of cancer-related deaths (2). Both of these cancer types are associated with a poor prognosis (1, 2). Despite the effectiveness of chemotherapy as a frontline cancer treatment, accumulating evidence from animal models suggests that chemotherapy may stimulate tumor growth and metastasis (3–22). The Révész effect, described in 1956, demonstrates that tumor cell death (“debris”) generated by cancer therapy, such as radiation, accelerates tumor engraftment (23). Follow-up studies have confirmed the Révész effect, whereby radiation-generated debris stimulates tumor growth via a proinflammatory response (24–29). Dead cell–derived mediators also stimulate tumor cell growth (30, 31). Notably, large numbers of cells are known to die in established tumors (32), which can lead to endogenous tumor-promoting debris in the tumor microenvironment (8, 33–35).Chemotherapy-generated tumor cell debris (e.g., apoptotic and necrotic cells) promotes tumor growth and metastasis via several mechanisms, including: 1) triggering a storm of proinflammatory and proangiogenic eicosanoids and cytokines (8, 9, 33, 35–38); 2) hijacking tumor-associated macrophages (TAMs) (37, 39); 3) inactivating M1-like TAMs (37); and 4) inducing immunosuppression and limiting antitumor immunity (40–42). Importantly, a metastatic phenotype and poor survival in cancer patients can be predicted by high levels of tumor cell debris (43–48). Thus, every attempt to induce tumor cell death is a double-edged sword as the resulting debris stimulates the growth of surviving tumor cells (8, 25, 33, 34, 35, 37, 38, 49–53). Tumor cells that survive treatment with chemotherapy or radiation undergo tumor cell repopulation (29). Yet, no strategy currently exists to stimulate the clearance or resolution of therapy-induced tumor cell debris and inflammation in cancer patients (35, 54).The failure to resolve inflammation-associated debris critically drives the pathogenesis of many human diseases, including cancer (8, 35, 55). Inflammation is regulated by a balance between inflammation-initiating eicosanoids (e.g., prostaglandins, leukotrienes, and thromboxanes) and specialized proresolving lipid autacoid mediators (SPMs; e.g., resolvins and lipoxins), which are endogenously produced in multiple tissues throughout the human body (56). Notably, arachidonic acid metabolites, collectively called eicosanoids, are potent mediators of inflammation and cancer metastasis (57, 58). Epoxyeicosatrienoic acids (EETs, also named EpETrEs), key eicosanoid regulators of angiogenesis, also stimulate inflammation resolution via macrophage-mediated phagocytosis of cell debris (59–64). Because EETs are rapidly metabolized by soluble epoxide hydrolase (sEH) to the less active dihydroxyeicosatrienoic acids (DiHETEs) (62), inhibition of sEH stabilizes EETs (62, 65). Indeed, sEH is a key therapeutic target for pain, as well as neurodegenerative and inflammatory diseases, including cancer (33, 35, 65–74). Thus, sEH regulates inflammatory responses (62). Importantly, sEH inhibition reduces the circulating levels and the expression of pancreatic mRNA of inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in experimental acute pancreatitis in mice (75). Chronic pancreatitis is essential for the induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice, suggesting that inflammation is a critical driver of pancreatic cancer (76, 77). Potent, selective inhibitors of sEH have been demonstrated to suppress human cancers (e.g., glioblastoma) and inflammation-induced carcinogenesis (67, 71). Similarly, inhibition of sEH can suppress inflammatory bowel disease-induced carcinogenesis and inflammation-associated pancreatic cancer (74, 78). In addition, a dual inhibitor of c-RAF and sEH suppresses chronic pancreatitis and murine pancreatic intraepithelial neoplasia in mutant K-Ras–initiated carcinogenesis (72, 73). Likewise, dual cyclooxygenase-2 (COX-2)/sEH inhibitors (e.g., PTUPB) potentiate the antitumor activity of chemotherapy and suppress primary tumor growth and metastasis via inflammation resolution (33, 35, 66, 70).Cancer therapy-induced debris can stimulate tumor growth and metastasis via prostaglandin E₂ (PGE₂) in the tumor microenvironment (25, 35, 79). PGE₂ exerts its biological activity via four G protein-coupled receptors: EP1, EP2, EP3, and EP4 (80). Among these, EP4 is upregulated in both tumor cells and immune cells (e.g., macrophages) and exhibits protumorigenic activity in many human malignancies (e.g., breast, prostate, colon, ovarian, and lung) by regulating angiogenesis, lymphangiogenesis, liver metastasis, and lymphatic metastasis (81–85). Interestingly, PGE₂ impairs macrophage phagocytosis of pathogens via EP4 receptor activation (86–88). Moreover, EP4 stimulates cancer proliferation, migration, invasion, and metastasis (89). EP4 gene silencing inhibits metastatic potential in vivo in preclinical models of breast, prostate, colon, and lung cancer (85, 90). Additionally, EP4 antagonists can suppress proinflammatory cytokines (e.g., C-C motif chemokine ligand 2 [CCL2], IL-6, and C-X-C chemokine motif 8 [CXCL8]), reduce inflammation-dependent bone metastasis, and diminish immunosuppression, while restoring antitumor immunity (91–93). In a clinical study, the EP4 antagonist E7046 increased the levels of T cells and tumor-infiltrating M2 macrophages in patients with advanced malignancies (94). Intriguingly, EP4 antagonists enhance the tumor response to chemotherapy by inducing extracellular vesicle-mediated clearance of cancer cells (95). Notably, EP4 antagonists reverse chemotherapy resistance or enhance immune-based therapies in various tumor types, including lymphoma, colorectal cancer, and lung cancer (80, 93, 96). Thus, targeting the EP4 receptor may be a strategy to suppress debris-stimulated tumor growth and metastasis.Here, we demonstrate that tumor cell debris generated by chemotherapy (e.g., gemcitabine) stimulates primary hepato-pancreatic cancer growth and metastasis when coinjected with a subthreshold (nontumorigenic) inoculum of tumor cells. Chemotherapy-generated debris upregulated sEH and EP4, which triggered a macrophage-derived storm of proinflammatory and proangiogenic mediators. Inhibitors of sEH and EP4 antagonists promoted inflammation resolution through macrophage phagocytosis of tumor cell debris and reduced proinflammatory eicosanoid and cytokine production in the tumor microenvironment. Altogether, our data show that the combined pharmacological abrogation of sEH and EP4 can prevent hepato-pancreatic cancer and metastatic progression.     

Hemolysis-induced lethality involves inflammasome activation by heme

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K⁺ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.Hemolysis, hemorrhage, and rhabdomyolysis cause the release of large amounts of hemoproteins to the extracellular space, which, once oxidized, release the heme moiety, a potentially harmful molecule due to its prooxidant, cytotoxic, and inflammatory effects (1, 2). Scavenging proteins such as haptoglobin and hemopexin bind hemoglobin and heme, respectively, promoting their clearance from the circulation and delivery to cells involved with heme catabolism. Heme oxygenase cleaves heme and generates equimolar amounts of biliverdin, carbon monoxide (CO) and iron (2). Studies using mice deficient for haptoglobin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1) demonstrate the importance of these proteins in controlling the deleterious effects of heme. Both Hp^−/− and Hx^−/− mice have increased renal damage after acute hemolysis induced by phenyhydrazine (Phz) compared with wild-type mice (3, 4). Mice lacking both proteins present splenomegaly and liver inflammation composed of several foci with leukocyte infiltration after intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascular function (6–8). Lack of heme oxygenase 1 (Hmox1^−/−) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9–15). This salutary effect of HO-1 has been attributed to its effect of reducing heme amounts as well as generating the cytoprotective molecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16, 17), inhibits neutrophil apoptosis (18), triggers cytokine and lipid mediator production by macrophages (19, 20), and increases the expression of adhesion molecules and tissue factor on endothelial cells (21–23). Heme cooperates with TNF, causing hepatocyte apoptosis in a mechanism dependent on reactive oxygen species (ROS) generation (12). Whereas heme-induced TNF production depends on functional toll-like receptor 4 (TLR4), ROS generation in response to heme is TLR4 independent (19). We recently observed that heme triggers receptor-interacting protein (RIP)1/3-dependent macrophage-programmed necrosis through the induction of TNF and ROS (15). The highly unstable nature of iron is considered critical for the ability of heme to generate ROS and to cause inflammation. ROS generated by heme has been mainly attributed to the Fenton reaction. However, recent studies suggest that heme can generate ROS through multiple sources, including NADPH oxidase and mitochondria (22, 24–27).Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28), but the mechanisms by which heme operates in different conditions are not completely understood. Blocking the prooxidant effects of heme protects cells from death and prevents tissue damage and lethality in models of malaria and sepsis (12, 13, 15). Importantly, two recent studies demonstrated the pathogenic role of heme-induced TLR4 activation in a mouse model of sickle cell disease (29, 30). These results highlight the great potential of understanding the molecular mechanisms of heme-induced inflammation and cell death as a way to identify new therapeutic targets.Hemolysis and heme synergize with microbial molecules for the induction of inflammatory cytokine production and inflammation in a mechanism dependent on ROS and Syk (24). Processing of pro–IL-1β is dependent on caspase-1 activity, requiring assembly of the inflammasome, a cytosolic multiprotein complex composed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (31–33). The most extensively studied inflammasome is the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). NLRP3 and pro–IL-1β expression are increased in innate immune cells primed with NF-κB inducers such as TLR agonists and TNF (34, 35). NLRP3 inflammasome is activated by several structurally nonrelated stimuli, such as endogenous and microbial molecules, pore-forming toxins, and particulate matter (34, 35). The activation of NLRP3 involves K⁺ efflux, increase of ROS and Syk phosphorylation. Importantly, critical roles of NLRP3 have been demonstrated in a vast number of diseases (34, 36). We hypothesize that heme causes the activation of the inflammasome and secretion of IL-1β. Here we found that heme triggered the processing and secretion of IL-1β dependently on NLRP3 inflammasome in vitro and in vivo. The activation of NLRP3 by heme was dependent on Syk, ROS, and K⁺ efflux, but independent of lysosomal leakage, ATP release, or cell death. Finally, our results indicated the critical role of macrophages, the NLRP3 inflammasome, and IL-1R to the lethality caused by sterile hemolysis.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Two-dimensional electronic–vibrational sum frequency spectroscopy for interactions of electronic and nuclear motions at interfaces

Interactions of electronic and vibrational degrees of freedom are essential for understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Here, we present the development of interface-specific two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy for electronic–vibrational couplings for excited states at interfaces and surfaces. We demonstrate this 2D-EVSFG technique by investigating photoexcited interface-active (E)-4-((4-(dihexylamino) phenyl)diazinyl)-1-methylpyridin-1- lum (AP3) molecules at the air–water interface as an example. Our 2D-EVSFG experiments show strong vibronic couplings of interfacial AP3 molecules upon photoexcitation and subsequent relaxation of a locally excited (LE) state. Time-dependent 2D-EVSFG experiments indicate that the relaxation of the LE state, S₂, is strongly coupled with two high-frequency modes of 1,529.1 and 1,568.1 cm⁻¹. Quantum chemistry calculations further verify that the strong vibronic couplings of the two vibrations promote the transition from the S₂ state to the lower excited state S₁. We believe that this development of 2D-EVSFG opens up an avenue of understanding excited-state dynamics related to interfaces and surfaces.

Electronic and vibrational degrees of freedom are the most important physical quantities in molecular systems at interfaces and surfaces. Knowledge of interactions between electronic and vibrational motions, namely electronic–vibrational couplings, is essential to understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Many excited-states relaxation processes occur at interfaces and surfaces, including charge transfer, energy transfer, proton transfer, proton-coupled electron transfer, configurational dynamics, and so on (1–11). These relaxation processes are intimately related to the electronic–vibrational couplings at interfaces and surfaces. Strong electronic–vibrational couplings could promote nonadiabatic evolution of excited potential energy and thus, facilitate chemical reactions or intramolecular structural changes of interfacial molecules (10, 12, 13). Furthermore, these interactions of electronic and vibrational degrees of freedom are subject to solvent environments (e.g., interfaces/surfaces with a restricted environment of unique physical and chemical properties) (9, 14, 15). Despite the importance of interactions of electronic and vibrational motions, little is known about excited-state electronic–vibrational couplings at interfaces and surfaces.Interface-specific electronic and vibrational spectroscopies enable us to characterize the electronic and vibrational structures separately. As interface-specific tools, second-order electronic sum frequency generation (ESFG) and vibrational sum frequency generation (VSFG) spectroscopies have been utilized for investigating molecular structure, orientational configurations, chemical reactions, chirality, static potential, environmental issues, and biological systems at interfaces and surfaces (16–52). Recently, structural dynamics at interfaces and surfaces have been explored using time-resolved ESFG and time-resolved VSFG with a visible pump or an infrared (IR) pump thanks to the development of ultrafast lasers (6–9, 13–15, 49, 53–61). Doubly resonant sum frequency generation (SFG) has been demonstrated to probe both electronic and vibration transitions of interfacial molecular monolayer (15, 62–71). This frequency-domain two-dimensional (2D) interface/surface spectroscopy could provide information regarding electronic–vibrational coupling of interfacial molecules. However, contributions from excited states are too weak to be probed due to large damping rates of vibrational states in excited states (62, 63). As such, the frequency-domain doubly resonant SFG is used only for electronic–vibrational coupling of electronic ground states. Ultrafast interface-specific electronic–vibrational spectroscopy could allow us to gain insights into how specific nuclear motions drive the relaxation of electronic excited states. Therefore, development of interface-specific electronic–vibrational spectroscopy for excited states is needed.In this work, we integrate the specificity of interfaces and surfaces into the capabilities of ultrafast 2D spectroscopy for dynamical electronic–vibrational couplings in excited states of molecules; 2D interface-specific spectroscopies are analogous to those 2D spectra in bulk that spread the information contained in a pump−probe spectrum over two frequency axes. Thus, one can better interpret congested one-dimensional signals. Two-dimensional vibrational sum frequency generation (2D-VSFG) spectroscopy was demonstrated a few year ago (72–74). Furthermore, heterodyne 2D-VSFG spectroscopy using middle infrared (mid-IR) pulse shaping and noncollinear geometry 2D-VSFG experiments have also been developed to study vibrational structures and dynamics at interfaces (31, 75–78). Recently, two-dimensional electronic sum frequency generation (2D-ESFG) spectroscopy has also been demonstrated for surfaces and interfaces (79). On the other hand, bulk two-dimensional electronic–vibrational (2D-EV) spectroscopy has been extensively used to investigate the electronic relaxation and energy transfer dynamics of molecules, biological systems, and nanomaterials (80–90). The 2D-EV technique not only provides electronic and vibrational interactions between excitons or different excited electronic states of systems but also, identifies fast nonradiative transitions through nuclear motions in molecules, aggregations, and nanomaterials. However, an interface-specific technique for two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy has yet to be developed.Here, we present the development of 2D-EVSFG spectroscopy for the couplings of electronic and nucleic motions at interfaces and surfaces. The purpose of developing 2D-EVSFG spectroscopy is to bridge the gap between the visible and IR regions to reveal how structural dynamics for photoexcited electronic states are coupled with vibrations at interfaces and surfaces. As an example, we applied this 2D-EVSFG experimental method to time evolution of electronic–vibrational couplings at excited states of interface-active molecules at the air–water interface.     

From the Cover: JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release.High-mobility group box 1 (HMGB1), a ubiquitous DNA-binding protein, is a promiscuous sensor driving nucleic acid-mediated immune responses and a pathogenic inflammatory mediator in sepsis, arthritis, colitis, and other disease syndromes (1–5). Immune cells actively release HMGB1 after activation by exposure to pathogen-associated molecular patterns or damage-associated molecular patterns, including lipopolysaccharide (LPS) and inflammasome agonists (1, 6, 7). High levels of extracellular HMGB1 accumulate in patients with infectious and sterile inflammatory diseases. Extracellular disulfide HMGB1 stimulates macrophages to release TNF and other inflammatory mediators by binding and signaling through Toll-like receptor (TLR)4. Reduced HMGB1 facilitates immune cell migration by interacting with the receptor for advanced glycation end products (RAGE) and CXCL12 (8–12), a process regulated by posttranslational redox-dependent mechanisms. Administration of neutralizing anti-HMGB1 mAbs or other HMGB1 antagonists significantly reduces the severity of inflammatory disease, promotes bacterial clearance during Pseudomonas aeruginosa or Salmonella typhimurium infection and attenuates memory impairment in sepsis survivors (1, 13–15). Together, these and other findings indicate the importance of a mechanistic understanding of HMGB1 release from activated immune cells and the regulatory signaling pathways controlling these processes.Most cytokines harbor a leader peptide that facilitates secretion through the endoplasmic reticulum–Golgi exocytotic route. HMGB1, which lacks a leader peptide, is released via unconventional protein secretion pathways (1, 6, 7). In quiescent cells, most HMGB1 is localized in the nucleus. Upon activation of immune cells, efficient HMGB1 release requires acetylation of HMGB1 within the two nuclear localization sequence (NLS) sites and subsequent HMGB1 accumulation in the cytoplasm (1, 6, 16–20). HMGB1 release is mediated by inflammasome activation during pyroptosis, a form of proinflammatory programmed cell death (6, 7, 22–24). Protein kinase (PK)R is a critical regulator of inflammasome-dependent HMGB1 release (6, 25). Pharmacological inhibition of PKR abrogates LPS-induced HMGB1 release by macrophages but does not prevent nuclear translocation of HMGB1 to cytoplasm. This suggests that some other, as yet unknown, inflammasome-independent pathway regulates HMGB1 translocation from nucleus to cytoplasm.We and others have previously established an important role of type 1 and type 2 interferons (IFNs) and downstream JAK/STAT1 signaling activation in mediating HMGB1 release (26–28). Pharmacological inhibition of JAK/STAT, genetic deletion of STAT1, or inhibition of extracellular IFN-β with neutralizing antibodies significantly abrogates LPS-induced HMGB1 release from macrophages (26–28). Importantly, pharmacological inhibition of the JAK/STAT1 pathway, genetic deletion of STAT1, or inhibition of IFN-β expression by genetic deletion of IRF3 significantly promotes survival in both lethal endotoxemia and experimental sepsis (28–30). Accordingly, we reasoned here that JAK/STAT1 may represent a critical signaling mechanism controlling HMGB1 translocation from nucleus to cytoplasm.     

Pop-out search instigates beta-gated feature selectivity enhancement across V4 layers

Visual search is a workhorse for investigating how attention interacts with processing of sensory information. Attentional selection has been linked to altered cortical sensory responses and feature preferences (i.e., tuning). However, attentional modulation of feature selectivity during search is largely unexplored. Here we map the spatiotemporal profile of feature selectivity during singleton search. Monkeys performed a search where a pop-out feature determined the target of attention. We recorded laminar neural responses from visual area V4. We first identified “feature columns” which showed preference for individual colors. In the unattended condition, feature columns were significantly more selective in superficial relative to middle and deep layers. Attending a stimulus increased selectivity in all layers but not equally. Feature selectivity increased most in the deep layers, leading to higher selectivity in extragranular layers as compared to the middle layer. This attention-induced enhancement was rhythmically gated in phase with the beta-band local field potential. Beta power dominated both extragranular laminar compartments, but current source density analysis pointed to an origin in superficial layers, specifically. While beta-band power was present regardless of attentional state, feature selectivity was only gated by beta in the attended condition. Neither the beta oscillation nor its gating of feature selectivity varied with microsaccade production. Importantly, beta modulation of neural activity predicted response times, suggesting a direct link between attentional gating and behavioral output. Together, these findings suggest beta-range synaptic activation in V4’s superficial layers rhythmically gates attentional enhancement of feature tuning in a way that affects the speed of attentional selection.

Throughout cortex, sensory information is organized into maps. This phenomenon is readily observable in visual cortex where maps organize information in both the radial (e.g., within cortical columns) and tangential (e.g., across a cortical area) dimensions (1–4). Importantly, sensory information attributed to these maps is malleable. For example, selective attention is linked to profound changes in neural activity organizing sensory information in both space and time (5–34).In visual cortex, cortical columnar microcircuits comprise many neurons that respond to the same location of visual space and similar stimulus features. For example, primary visual cortex (V1) features “orientation columns” consisting of neurons sharing response preference for the same stimulus orientation (35, 36) and “ocular dominance columns” consisting of neurons that preferentially respond to the same eye (37). Similar columnar organization for feature selectivity has been described across many other visual cortical areas, including area V2 (36, 38, 39), area V3 (40), middle temporal area (area MT) (41–43), and inferotemporal cortex (44–46). Midlevel visual cortical area V4, a well-studied area contributing to attentional modulation, follows suit with columnar organization of visual responses and feature preferences (44, 47–53). Yet, we do not know the extent to which attention impacts feature preferences along columns. While canonical microcircuit models of cortex predict laminar differences for attentional modulation [e.g., feedback-recipient extragranular layers modulating before granular layers (54–56)], how this modulation interacts with columnar feature selectivity is largely unknown.We sought to determine the spatiotemporal profile of feature preferences within the V4 laminar microcircuit during attentional selection. To address this question, we performed neurophysiological recordings along V4 layers in monkeys performing an attention-demanding pop-out search task. We identified feature columns demonstrating homogeneous feature preference along cortical depth. When the search array item presented in the column’s receptive field (RF) was unattended, the upper cortical layers were most selective. However, when attended, feature selectivity in the deep layers enhanced the most, resulting in overall strongest feature selectivity in both extragranular compartments. We further found that the enhancement of feature selectivity associated with attention was rhythmically gated in the beta range. While beta activity was measurable across both unattended and attended conditions, rhythmic gating of feature selectivity was only present with attention. Moreover, beta power modulating the neural response was predictive of response time (RT), suggesting a link between attentional gating and behavior. Synaptic currents revealed the beta rhythm originates in superficial cortical layers, which is compatible with top-down influence.     

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia–neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K⁺ concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30–100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.Microglia are tissue-resident macrophages in the CNS that become activated in most brain disorders, such as bacterial meningoencephalitis, multiple sclerosis, and Alzheimer’s disease (1, 2). Activation of microglia features changes in morphology and receptor expression, antigen presentation, cytokine release, migration, and phagocytosis, and it ranges from proinflammatory and potentially neurotoxic to anti-inflammatory and neuroprotective phenotypes (1, 3, 4). The mechanisms that control the transition of microglia to reactive phenotypes, including the impact on neuronal function, are mostly unknown, however (5–7).Sensing of microbial or modified endogenous ligands by microglia is mediated by innate pattern recognition receptors, such as scavenger receptors and Toll-like receptors (TLRs). A prime example is TLR4, which acts with CD14, MD-2, and lipopolysaccharide (LPS)-binding protein in recognizing LPS, a cell wall component of Gram-negative bacteria (8, 9). TLR4 is also central to microglial recognition of amyloid-β peptide, which is thought to be part of the inflammatory response in Alzheimer’s disease (7, 10).LPS has been widely used to study the molecular mechanisms of microglial activation in inflammatory neurodegeneration (1–3). In primary monocultures and microglia-neuron cultures, LPS exposure alone or in combination with IFN-γ for a “booster” triggers the massive release of proinflammatory and cytotoxic factors, such as TNF-α, IL-6, and nitric oxide (NO), finally resulting in neuronal death (8, 11–18). Similar effects were observed in vivo after intracerebral administration of LPS (19–21). These and other studies have contributed to the concept that microglial TLR4 activation with LPS (i.e., with a single pathogenic stimulus) is sufficient to induce neurodegeneration (22, 23); however, this concept is biologically risky, and has been questioned in some experimental works and reviews (2–4, 11, 24, 25).Most previous studies focused on two aspects of microglial TLR4 activation with LPS: (i) the properties of the reactive microglial phenotype(s) and (ii) the degree of neurodegeneration. For this purpose, either simple culture systems or in vivo models, in which interactions with leukocytes infiltrating from the blood are inevitable, have been used (1, 4). Thus, it is widely unknown how TLR4 and IFN-γ receptor signaling in microglia individually contribute to neurotoxicity and neurodegeneration in situ. This aspect is highly relevant for several neurologic and psychiatric disorders. Moreover, concomitant alterations in neuronal information processing (i.e., dysfunction in excitatory pyramidal cells and inhibitory GABAergic interneurons, including astrocytes) have been little explored (25–27).We rigorously addressed these fundamental questions in postnatal neuronal tissue (1, 4). To mimic microglial confrontation with LPS in situ and, notably, in the absence of infiltrating leukocytes, we used organotypic hippocampal slice cultures that feature highly preserved cytoarchitectures and complex neuronal network functions (5, 28). Microglial interaction with infiltrating T helper type 1 (Th1) cells and/or natural killer (NK) cells was mimicked by recombinant IFN-γ administration.     

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

Many pathogenic or symbiotic bacteria for plants or animals have evolved specialized molecular machines known as type III protein secretion systems (T3SSs) (1–3). These machines inject bacterially encoded effector proteins into target eukaryotic cells to modulate cellular processes and ensure the survival and replication of the pathogens or symbionts that encode them (4–6). Although the structural organization of this secretion machine has been largely derived from studies of the T3SSs of the bacterial pathogens Salmonella Typhimurium and Shigella flexneri (7–15), given the conservation of its core components, it is predicted that the T3SSs in other bacteria exhibit a similar architecture. The secretion machine itself is composed of a ∼5-Md multiprotein assembly known as the injectisome (1, 7, 14, 16, 17). This core structure consists of two large substructures, an envelope-associated needle complex (7, 16, 18, 19) and a large cytoplasmic complex known as the “sorting platform” (14, 15, 20).The needle complex is composed of a multiring hollow base, which anchors the injectisome to the bacterial envelope (18). The base encloses the export apparatus, a helical structure that serves as a conduit for the passage through the inner membrane of the proteins destined to transit this secretion pathway (21). The base is also linked to a needle-like filament, which protrudes several nanometers from the bacterial surface and is formed by a single protein arranged in a helical fashion (17, 22). The needle filament, which is traversed in its entire length by a central channel ∼3 nm in diameter, is linked to the base through a structure known as the inner rod (11) and is capped at its distal end by another substructure known as the “tip complex” (23, 24).The sorting platform is located in its entirety within the cytoplasm (14, 15, 20). It is made of six pods that form a cage-like enclosure, which is capped on its cytoplasmic side by a wheel-like structure that holds a hexameric ATPase. Also harbored within the sorting platform cage is the large cytoplasmic domain of one of the components of the export apparatus, which is arranged as two concentric rings and forms a conduit for the secreted substrates to reach the entrance of the export channel (25).A distinctive feature of the T3SSs is that their activation requires contact with the target eukaryotic cell (26, 27). The activation of the T3SS is followed by the deployment of the translocon substructure, which firmly anchors the injectisome to the target cell and serves as the passageway for the effector proteins across the eukaryotic cell membrane. Although little is known about the activation process, it is thought that sensing of the target cell by the tip complex initiates a signaling event that is transduced to the secretion machine by the needle filament itself (26–31). Activation of the secretion machine is then followed by the deployment of the translocon on the target cell membrane, which along with the tip complex and the needle filament, form a continuous passageway through which effector proteins transit from the bacterial cytoplasm to the cytosol of the eukaryotic cell (23, 32, 33). The composition of the tip complex has been the subject of some controversy. While it has been proposed that in the T3SSs of Yersinia spp., Pseudomonas aeruginosa, and Salmonella spp. the tip structure is made up of a single protein, LcrV (24), PcrV (34), and SipD (35), respectively, in the case of Shigella spp., it has been alternatively proposed to be composed of two proteins, IpaB and IpaD (36), or just IpaD (37, 38). The crystal structures of monomeric SipD and close homologs show that these proteins are arranged in three domains: an N-terminal α-helical hairpin, a central coiled-coil, and a mixed α/β carboxyl-terminal domain (39–42). It has been proposed that the N-terminal α-helical hairpin domain functions as a self-chaperone that prevents the self-oligomerization and/or the premature interaction of the tip protein with the needle filament subunit within the bacterial cytoplasm (39). A current hypothesis is that during assembly at the tip of the needle, the N-terminal α-helical hairpin of SipD/IpaD is displaced to allow other domains to interact with the needle. However, there is no structural information of the fully assembled tip complex to support this hypothesis. How the tip protein assembles into the tip complex, and how it is anchored at the distal end of the needle filament, is currently unknown in large part because of the absence of a high-resolution structure of this complex. Understanding of the events that lead to the activation of the secretion machine requires detailed knowledge not only of the structure of the tip complex that caps the needle filament but, importantly, its interface with the needle filament itself.Advances in cryoelectron microscopy (cryo-EM) have allowed the visualization of most components of the T3SS machine at high resolution, both in isolation as well as in situ (7–15). However, the tip structure has eluded high-resolution visualization, in part because existing needle complex isolation protocols result in the dissociation of the tip complex from the needle filament. Here we report the visualization at high resolution by cryo-EM of the tip structure of the needle complex of the S. Typhimurium T3SS encoded within its pathogenicity island 1. Combined with functional analysis, the structure provides major insight into the potential mechanisms of injectisome assembly and activation and fills one of the remaining gaps in the quest for the high-resolution visualization of the entire T3SS injectisome.