Homeostatic regulation of T follicular helper and antibody response to particle antigens by IL-1Ra of medullary sinus macrophage origin

Hepatitis B virus (HBV) vaccines are composed of surface antigen HBsAg that spontaneously assembles into subviral particles. Factors that impede its humoral immunity in 5% to 10% of vaccinees remain elusive. Here, we showed that the low-level interleukin-1 receptor antagonist (IL-1Ra) can predict antibody protection both in mice and humans. Mechanistically, murine IL-1Ra–inhibited T follicular helper (Tfh) cell expansion and subsequent germinal center (GC)-dependent humoral immunity, resulting in significantly weakened protection against the HBV challenge. Compared to soluble antigens, HBsAg particle antigen displayed a unique capture/uptake and innate immune activation, including IL-1Ra expression, preferably of medullary sinus macrophages. In humans, a unique polymorphism in the RelA/p65 binding site of IL-1Ra enhancer associated IL-1Ra levels with ethnicity-dependent vaccination outcome. Therefore, the differential IL-1Ra response to particle antigens probably creates a suppressive milieu for Tfh/GC development, and neutralization of IL-1Ra would resurrect antibody response in HBV vaccine nonresponders.

Follicular helper T (Tfh) cells are antigen-experienced CD4⁺ T cells within B cell follicles of secondary lymphoid organs, such as lymph nodes (LN), spleens, and Peyer’s patches, that constitutively express the B cell follicle homing receptor CXCR5 (1). Upon cellular interaction and cross-signaling with their cognate follicular B (FoB) cells in the presence of follicular dendritic cells (FDCs), Tfh cells trigger the formation and maintenance of germinal centers (GCs) through the expression of CD40 ligand and the secretion of IL-21 and IL-4 (2–4). Tfh-dependent paracrine activation of CD40 results in B cell survival and differentiation in the GC (5), whereas isotype class switching is believed to occur predominantly outside GCs. Therefore, Tfh cells play a critical role in mediating the selection of high-affinity B cells that differentiate either into plasma cells or memory B cells (6–11).Besides the inducible T cell costimulator (ICOS) that activates Tfh cells to secrete IL-21, other cytokines [e.g., IL-2 (12), IL-6 (13), and IL-7 (14)] also signal for Tfh cell differentiation. The role of IL-1 signaling remained puzzling until recently: Tfh cells can be primed by IL-1β, whose production is licensed by IFN-β in response to infectious agents (15). Such featured innate response of IFN-β and IL-1β relies on the activation of TLR and inflammasomes by live, but not dead, bacteria or recombinant vaccines (16, 17). Therefore, OVA antigen augments Tfh cell response in mice only when IL-1β is exogenously applied at a nonphysiological high concentration (18), whereas endogenous IL-1β/IL-1R1 signaling may not be required for antibody responses to T-dependent or -independent antigens (19–21). We reasoned that IL-1Ra (encoded by IL-1rn), which can compete with IL-1 for binding to IL-1R1 in the homeostatic inflammatory response (22–24), would intrinsically antagonize IL-1β/IL-1R1 signaling for Tfh/GC development. For example, IL-1rn^−/− mice exhibit an excessive antibody response to sheep red blood cells immunization (25, 26). A thorough investigation is required to dissect how IL-1 and IL-1Ra mutually regulate a homeostatic Tfh/GC response.LN macrophages are conventionally divided into two subtypes. Subcapsular sinus (SCS, CD169⁺F4/80⁻) macrophages are specialized antigen presenting cells that capture certain particle or opsonized antigens and display them intact for cognate recognition by FoB cells (27–30). SCS macrophages also relay immune complex to noncognate B cells for antibody affinity maturation (30). Macrophages in medullary sinus (MSM, CD169⁺F4/80⁺), in contrast, are potent in phagocytosis (31) for clearance of pathogens and particulate antigens from the lymph. It has been postulated 10 y ago that SCS may capture particle antigens, such as hepatitis B virus (HBV) vaccine, and migrate to follicles to facilitate more effective activation of B cells and FDCs (32). In this work, we found that murine antibody response inversely correlated to IL-1Ra level and clearly distinguished responders from nonresponders in volunteers receiving HBV vaccination. Further studies showed that LN macrophages subsets exhibited different capture and activation kinetics for particle and soluble antigens, and IL-1Ra expression by MSM could critically modulate IL-1R1 potentiation of Tfh cells and, hence, the specific antibody response to particle antigens. Therefore, mice lacking IL-1Ra or with IL-1Ra being neutralized yielded more robust antibody response to HBV vaccine and enables protection against chronic HBV infection.     

Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Parkinson’s disease (PD) is characterized by accumulation of α-synuclein (αSyn; encoded by the SNCA gene) (1). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal cell death in part via microglial activation (2, 3). Moreover, misfolded proteins in general are thought to interact with brain microglia, triggering microglial activation that contributes to neurodegenerative disorders, although microglial phagocytosis may also initially clear aberrant proteins to afford some degree of protection (2, 4). Additionally, in Alzheimer’s disease (AD), amyloid-β peptide (Aβ) is thought to trigger similar processes in microglia (5–7); however, the mechanism for this trigger is still poorly understood.Microglial cells contribute to neuroinflammation, specifically that mediated by the inflammasome. In particular, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with several neurodegenerative disorders, although other types of inflammation may also be important in this regard (8). The NLRP3 inflammasome is a multiprotein complex that responds to cell stress and pathogenic stimuli to promote activation of caspase-1, which in turn mediates maturation and release of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18 (9–11). NLRP3 inflammasome activation is a two-step process, involving an initial priming step and a secondary trigger. Priming involves a proinflammatory stimulus, such as endotoxin, a ligand for Toll-like receptor 4 (TLR4), that increases the abundance of NLRP3 and promotes de novo synthesis of pro–IL-1β via nuclear factor κB (11). The secondary trigger promotes inflammasome complex assembly and caspase-1 activation that in turn mediates the cleavage of pro–IL-1β and subsequent release of mature IL-1β. There are various secondary triggers, including adenosine triphosphate (ATP), microparticles, and bacterial toxins, all of which somehow lead to mitochondrial damage and release of oxidized mitochondrial DNA (11). Neuroinflammation has been reported in both human PD and AD brains (12–15), and NLRP3 inflammasome activation in particular has been observed in mouse models of PD and AD (7, 16). Importantly, in these PD models, dopaminergic (DA) neurons in the substantia nigra are resistant to damage in NLRP3-deficient mice compared with wild-type (WT) mice (16). Interestingly, a recent report identified an NLRP3 polymorphism that confers decreased risk in PD (17). Several groups have reported that fibrillar αSyn can activate the NLRP3 inflammasome in mice and in human monocytes (18–22), but it remains unknown if human brain microglia can be activated in this manner. Critically, antibodies targeting misfolded proteins are being tested in human clinical trials for several neurodegenerative diseases, including AD and PD; however, it is still unclear how antibodies to αSyn might affect this inflammatory response. In this study, we characterized the response of human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) to oligomeric/fibrillar αSyn in vitro and in vivo, using engraftment of hiMG in humanized mice. We used these immunocompromised mice because they prevent human cell rejection and express three human genes that support human cell engraftment (23). We show that αSyn and, even more so, αSyn–antibody complexes activate the NLRP3 inflammasome. Moreover, this process is further sensitized by the presence of Aβ and its cognate antibodies. These observations are of heightened interest because recent studies have shown that both misfolded Aβ and αSyn are present in several neurodegenerative disorders such as AD and Lewy body dementia (LBD), a form of dementia that can occur in the setting of PD (24–26).     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.     

A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aβ fibers. However, the mechanism by which Aβ fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP⁺) and show that specific ablation or silencing of AAV-NpyP⁺ SDH interneurons converted touch-sensing Aβ fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP⁺ neurons received excitatory inputs from Aβ fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP⁺ neurons exhibited deeper resting membrane potentials, and their excitation by Aβ fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP⁺ neurons in nerve-injured rats reversed Aβ fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aβ fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

Damage to the nervous system by cancer, diabetes, chemotherapy, infection, or traumatic injury causes neuropathic pain, a highly debilitating chronic pain condition (1). A cardinal symptom of neuropathic pain is mechanical allodynia, pain that is produced by innocuous mechanical stimulus, such as light touch. The mechanisms underlying mechanical allodynia are poorly understood. Currently available treatments including opioids are largely ineffective.Light mechanical information from the skin is conveyed to the spinal dorsal horn (SDH) via primary afferent low-threshold mechanoreceptors (LTMRs), such as Aβ fibers. These LTMRs are considered to mediate mechanical allodynia (2–7). A major question is where and how touch signals are pathologically converted to pain in the context of nerve damage. One potential region could be the SDH where Aβ fibers and nociceptors interact through interneurons (6, 8–10), as depicted in the gate control theory of pain (11). Over the last 5 y, studies using multiple lines of transgenic mice have identified several subsets of excitatory and inhibitory interneurons in the SDH that are genetically defined (12–18) and shown that these subsets are involved in peripheral nerve injury (PNI)-induced mechanical hypersensitivity (assessed using von Frey filaments). However, the behavioral hypersensitivity by these filaments involves activation not only of LTMRs but also of nociceptors (19–24) and is effectively suppressed by treatment with morphine (18, 25). Thus, the mechanisms underlying LTMR-derived and morphine-resistant neuropathic allodynia are still poorly understood.Using a transgenic rat line W-TChR2V4 in which channelrhodopsin-2 (ChR2) was expressed at nerve endings associated with Merkel cells and lamellar cells to form Meissner’s corpuscle-like structures in the skin (26), we recently reported that following PNI, stimulation of touch-sensing Aβ fibers by illuminating the rats with blue light elicited morphine-resistant mechanical allodynia-like responses (27). Furthermore, Aβ fiber stimulation after PNI causes activation of lamina I neurons, which are normally silent in response to this stimulation. This raises the possibility that alterations in SDH circuits after PNI underscore the conversion of Aβ fiber-derived signals to morphine-resistant pain, but the underlying mechanisms remain to be determined. Considering previous findings (6, 8–11), a possible mechanism for the conversion might involve a loss or reduction of the activity of inhibitory interneurons in the SDH. A single-cell RNA sequencing study has shown that SDH inhibitory interneurons are genetically divided into over 10 subsets, some of which express mRNA encoding neuropeptide Y (NPY) (28). In immunohistochemistry, NPY has been shown to be expressed in inhibitory interneurons (29, 30). Furthermore, previous studies have demonstrated that spinal NPY has inhibitory effects on chronic pain, including PNI-induced mechanical hypersensitivity (31, 32). Thus, to identify a subset of inhibitory SDH interneurons that contributes to the behavioral symptom evoked by optical stimulation of the primary afferent Aβ fibers in the W-TChR2V4 rats after PNI, we focused on the role of inhibitory SDH interneurons operated by an adeno-associated viral (AAV) vector including a Npy promoter (AAV-NpyP). Using the optogenetic approach for neuropathic allodynia (27) combined with chemogenetics, electrophysiology, and transsynaptic tracing, this study reveals that diminished inhibitory tone of these SDH neurons operated by AAV-NpyP contributes to Aβ fiber-derived neuropathic pain-like behavioral responses. Our findings suggest that this subset of neurons could be a therapeutic target for treating neuropathic mechanical allodynia.     

Impact of prenatal maternal cytokine exposure on sex differences in brain circuitry regulating stress in offspring 45 years later

Stress is associated with numerous chronic diseases, beginning in fetal development with in utero exposures (prenatal stress) impacting offspring’s risk for disorders later in life. In previous studies, we demonstrated adverse maternal in utero immune activity on sex differences in offspring neurodevelopment at age seven and adult risk for major depression and psychoses. Here, we hypothesized that in utero exposure to maternal proinflammatory cytokines has sex-dependent effects on specific brain circuitry regulating stress and immune function in the offspring that are retained across the lifespan. Using a unique prenatal cohort, we tested this hypothesis in 80 adult offspring, equally divided by sex, followed from in utero development to midlife. Functional MRI results showed that exposure to proinflammatory cytokines in utero was significantly associated with sex differences in brain activity and connectivity during response to negative stressful stimuli 45 y later. Lower maternal TNF-α levels were significantly associated with higher hypothalamic activity in both sexes and higher functional connectivity between hypothalamus and anterior cingulate only in men. Higher prenatal levels of IL-6 were significantly associated with higher hippocampal activity in women alone. When examined in relation to the anti-inflammatory effects of IL-10, the ratio TNF-α:IL-10 was associated with sex-dependent effects on hippocampal activity and functional connectivity with the hypothalamus. Collectively, results suggested that adverse levels of maternal in utero proinflammatory cytokines and the balance of pro- to anti-inflammatory cytokines impact brain development of offspring in a sexually dimorphic manner that persists across the lifespan.

Repeated and prolonged adverse responses to negative stress have been associated with increased risk for many chronic diseases, including psychiatric and cardiovascular disorders. In fact, perturbations in the in utero development of the stress response circuitry have played a key role underlying the developmental origins of disease, including what has been termed prenatal-stress models of chronic disease (1–6). These in utero perturbations may also contribute to sex differences in disease risk (2, 4, 6–10), given that the brain circuitry regulating the stress response contains some of the most highly sexually dimorphic regions in the brain (2, 4, 9, 11). That is, they develop differently in the male and female brain in utero, and when developmentally disrupted, have long-lasting effects on sex-dependent disease risk (5).Brain circuitry involved in the stress response system includes arousal in the hypothalamus (HYPO), amygdala (AMYG), and periaqueductal gray (PAG) and inhibitory control of arousal by the medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC), and hippocampus (HIPP). These regions not only regulate response to stress but also steroid hormone physiology through the hypothalamic-pituitary-adrenal (HPA) and -gonadal axes. Primary coactivators of the HPA axis are proinflammatory cytokines, that is, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6. Receptors for these cytokines are located contiguously with glucocorticoid and gonadal hormone receptors in brain regions that regulate stress response circuitry and are densest in the paraventricular nucleus (PVN) of the HYPO and HIPP.Thus, the HPA axis is a critical junction for immune factors and steroid hormones, and as a result, the regulation of stress responses (2, 4, 6–8, 10, 12). The release of maternal immune molecules (e.g., TNF-α, IL-1β and IL-6) (4, 5, 13, 14) and glucocorticoids (13, 15, 16) can impact fetal development by stimulating placental production of corticotropin-releasing hormone (CRH) and thereby impact HPA-axis function in the fetus (17). The release of glucocorticoids (i.e., cortisol) has inhibitory effects on cytokine release of TNF-α, IL-1β, and IL-6, with TNF-α being most sensitive to this negative feedback loop (18). This chain of events is thought to have long-term consequences for the offspring’s brain health. Preclinical studies have shown neurologic changes in offspring induced by maternal immune activation that manifest as behavioral deficits in spatial learning and memory (14, 19, 20), increased anxiety and depressive behaviors (21), neurobiologic dysregulation, including hypomyelination and reduced neurogenesis (22, 23), and biochemical dysfunction expressed as decreases in serotonin (24) and alterations in dopaminergic markers (25). Sex differences in offspring outcomes depended on timing of the adverse prenatal maternal immune exposure. Earlier in utero, adverse exposures had greater impact on male offspring and later in utero and postnatal exposure on female offspring (26, 27). Further evidence for this comes from clinical studies of the effects of maternal prenatal immune compromise on offspring risk for sex differences in psychiatric and neurodevelopmental outcomes, for example, from ours (5, 8, 28, 29) and others’ (6, 19, 30).Despite evidence from preclinical and human studies that maternal immune activity is associated with sex differences in stress-mediated conditions in offspring, it remains to be shown how brain circuitry in adults is impacted by prior exposure to maternal immune activity in utero and the extent to which this impact is sex dependent. Here, we had the unique opportunity to investigate this in the context of a pregnancy cohort with follow-up of offspring in middle adulthood. We investigated whether concentrations of in utero proinflammatory cytokines in maternal sera (drawn at the start of the third trimester) are associated with sex differences in activity in specific brain regions that regulate stress and immune function 45 y later. We predicted that adverse concentrations of proinflammatory cytokines would be associated with hyperactivity in stress-arousal regions (in particular, HYPO) and hypoactivity in inhibitory regions of the stress response (in particular, HIPP), assessed by functional MRI (fMRI). Our rationale was, in part, based on the fact that HIPP has a negative feedback role on HYPO, specifically PVN, and PVN and HIPP are brain regions most dense with receptors for TNF-α, IL-1β, and IL-6 (31, 32).     

Targeting tumor-derived NLRP3 reduces melanoma progression by limiting MDSCs expansion

Interleukin-1β (IL-1β)–mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1β production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1β expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1β (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8⁺ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti–PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti–PD-1 therapy.

Tumorigenesis is initiated by genomic alterations, leading to cell transformation, proliferation, and resistance to apoptotic signals, which ultimately lead to metastasis and tissue invasion. Tumor progression is also linked to dysregulated inflammation, which is characterized by cytokine signaling between cancer and noncancer cells (1, 2). The proinflammatory cytokine interleukin-1β (IL-1β) mediates several inflammatory diseases and is a pivotal cytokine in initiating inflammatory responses (3). In the context of malignancy, IL-1β is a validated target in mouse models of cancer, including melanoma, where the cytokine contributes to immunosuppression, angiogenesis, metastasis, and regulation of myeloid-derived suppressor cells (MDSCs) (1, 2, 4, 5). In humans, IL-1β is overexpressed in biopsies from metastatic melanoma patients, suggesting a possible role in the melanoma-induced inflammation (6).Processing of IL-1β is largely governed by inflammasomes, cytosolic macromolecular complexes responsible for the conversion of biologically inactive IL-1β and IL-18 precursors into their active forms via caspase-1 cleavage (7). The nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) is the most studied of the inflammasome sensors driving IL-1β–mediated conditions from sterile inflammation to rare hereditary syndromes (8, 9). NLRP3 is particularly relevant to the processing of IL-1β in melanoma because NLRP3 is constitutively expressed in melanoma cell lines (6) and NLRP3 polymorphisms are linked to increased risk to develop melanoma (10). Whereas these studies indicate a possible role for NLRP3 in melanoma progression, the biological function for NLRP3 in melanoma remains unclear. Furthermore, although it is well known that inflammation participates in the development and progression of melanoma (11–13), the inflammatory pathways that drive this process are still poorly characterized and no therapy developed to date is actually designed to specifically target inflammatory pathways in melanoma. Here, using genetic models of NLRP3 depletion and a specific pharmacological inhibitor of NLRP3 (14), we show that NLRP3 represents a melanoma intrinsic pathway exploited for tumor-mediated immune escape. We demonstrate that tumor-derived NLRP3 activation induces MDSC expansion, which suppresses recruitment and activation of antitumor immunity.From a clinical standpoint, immune checkpoint therapy (ICT) has significantly improved the outcome for melanoma patients, and numerous studies have demonstrated that expression of PD-1/PD-L1 and CTLA4 are often predictors for efficacy of immunotherapy. However, the number of patients that are unresponsive to ICT or relapse continues to rise, and clinical data show that expression of immune checkpoints do not always correlate with responses (15). The limited response to monotherapy in some patients suggests that intrinsic pathways in melanoma cells, such as expression of checkpoint ligands, are not the only mechanisms that drive tumor progression. Identification of other tumor-specific strategies provides an opportunity to interrupt the oncogenic process and improve survival in this population. For example, melanoma-associated inflammation facilitates tumor progression (11, 16) and, specifically, is linked to IL-1β activity (4, 17). An approach for reducing IL-1β activity is via inhibition of NLRP3. Here, we show that disruption of the NLRP3 signaling in combination with ICT increases antitumor activity. The data support the concept that tumor NLRP3 activation represents an intrinsic pathway that favors tumor immune escape. Thus, targeting NLRP3 represents an innovative strategy for treating melanoma, especially in the context of immunotherapy resistance tumors.     

Addiction to Golgi-resident PI4P synthesis in chromosome 1q21.3–amplified lung adenocarcinoma cells

A chromosome 1q21.3 region that is frequently amplified in diverse cancer types encodes phosphatidylinositol (PI)-4 kinase IIIβ (PI4KIIIβ), a key regulator of secretory vesicle biogenesis and trafficking. Chromosome 1q21.3–amplified lung adenocarcinoma (1q-LUAD) cells rely on PI4KIIIβ for Golgi-resident PI-4-phosphate (PI4P) synthesis, prosurvival effector protein secretion, and cell viability. Here, we show that 1q-LUAD cells subjected to prolonged PI4KIIIβ antagonist treatment acquire tolerance by activating an miR-218-5p–dependent competing endogenous RNA network that up-regulates PI4KIIα, which provides an alternative source of Golgi-resident PI4P that maintains prosurvival effector protein secretion and cell viability. These findings demonstrate an addiction to Golgi-resident PI4P synthesis in a genetically defined subset of cancers.

The term “oncogene addiction” was coined to describe cancer cells’ exquisite dependence on individual oncogenes to sustain the malignant phenotype (1, 2). Examples include the BCR-ABL oncogene produced by a chromosome 9:22 translocation in chronic myelogenous leukemia and the somatically mutated EGFR oncogene in lung adenocarcinoma (LUAD) (3–5). In both cancer types, the mutant kinases are bona fide oncogenes in vitro and in vivo (6, 7). Although patients treated with selective kinase inhibitors attain profound clinical responses (8), chronic exposure of patients to targeted therapeutics is followed by disease relapse owing to an almost universal reactivation of mutant kinase activity, demonstrating that most cancers retain an underlying addiction to oncogene-induced signaling pathways (2, 9). Elucidating the molecular underpinnings of oncogene reactivation may lead to improved therapeutic strategies.Heightened secretion of protumorigenic effector proteins promotes metastasis and acquired resistance to targeted therapeutics (10, 11). The conventional secretory pathway directs the transport of secretory vesicles from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus (12). Tensile forces exerted on Golgi membranes activate secretory vesicle biogenesis and are mediated by a Golgi phosphoprotein-3 (GOLPH3)/F-actin protein complex (13). GOLPH3 binds to phosphatidylinositol (PI)-4-phosphate (PI4P), which tethers GOLPH3 to Golgi membranes and is generated by the Golgi-resident PI-4 kinases PI4KIIα and PI4KIIIβ (10, 13, 14).A chromosome 1q21.3 region that is frequently amplified in diverse cancer types encodes PI4KIIIβ (11, 15). Chromosome 1q21.3–amplified LUAD (1q-LUAD) cells undergo apoptosis following treatment with small-molecule PI4KIIIβ antagonists or depletion of PI4KIIIβ–dependent secreted proteins (11), establishing that PI4KIIIβ–dependent secreted proteins are prosurvival effectors in 1q-LUAD cells. On the basis of this conceptual framework, here, we postulated that chromosome 1q21.3 amplifications confer an addiction to Golgi-resident PI-4 kinase activity.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.