High-dimensional single-cell analysis of human natural killer cell heterogeneity.

Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.

Incidence of radiographic and clinically significant pneumothorax or hemothorax after thoracic discectomy via mini-open lateral retropleural approach without prophylactic chest tube placement.

OBJECTIVE: The mini-open lateral retropleural (MO-LRP) approach is an effective option for surgically treating thoracic disc herniations, but the approach raises concerns for pneumothorax (PTX). However, chest tube placement causes insertion site tenderness, necessitates consultation services, increases radiation exposure (requires multiple radiographs), delays the progression of care, and increases narcotic requirements. This study examined the incidence of radiographic and clinically significant PTX and hemothorax (HTX) after the MO-LRP approach, without the placement of a prophylactic chest tube, for thoracic disc herniation. METHODS: This study was a single-institution retrospective evaluation of consecutive cases from 2017 to 2022. Electronic medical records were reviewed, including postoperative chest radiographs, radiology and operative reports, and postoperative notes. The presence of PTX or HTX was determined on chest radiographs obtained in all patients immediately after surgery, with interval radiographs if either was present. The size was categorized as large (≥ 3 cm) or small (< 3 cm) based on guidelines of the American College of Chest Physicians. PTX or HTX was considered clinically significant if it required intervention. RESULTS: Thirty patients underwent thoracic discectomy via the MO-LRP approach. All patients were included. Twenty patients were men (67%), and 10 (33%) were women. The patients ranged in age from 25 to 74 years. The most commonly treated level was T11-12 (n = 11, 37%). Intraoperative violation of parietal pleura occurred in 5 patients (17%). No patient had prophylactic chest tube placement. Fifteen patients (50%) had PTX on postoperative chest radiographs; 2 patients had large PTXs, and 13 had small PTXs. Both patients with large PTXs had expansion on repeat radiographs and were treated with chest tube insertion. Of the 13 patients with a small PTX, 1 required 100% oxygen using a nonrebreather mask; the remainder were asymptomatic. One patient, who had no abnormal findings on the immediate postoperative chest radiograph, developed an incidental HTX on postoperative day 6 and was treated with chest tube insertion. Thus, 3 patients (10%) required a chest tube: 2 for expanding PTX and 1 for delayed HTX. CONCLUSIONS: Most patients who undergo thoracic discectomy via the MO-LRP approach do not develop clinically significant PTX or HTX. PTX and HTX in this patient population should be treated with a chest tube only when there are postoperative clinical and radiographic indications.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Reciprocal Changes in Sagittal Spinal Alignment After L5-S1 Anterior Lumbar Interbody Fusion.

OBJECTIVE: Degenerative diseases of the lumbar spine decrease lumbar lordosis (LL). Anterior lumbar interbody fusion (ALIF) at the L5-S1 disc space improves segmental lordosis, LL, and sagittal balance. This study investigated reciprocal changes in spinopelvic alignment after L5-S1 ALIF. METHODS: A retrospective chart review identified patients who underwent L5-S1 ALIF with or without posterior fixation at a single institution (November 1, 2016 to October 1, 2021). Changes in pelvic tilt, sacral slope, proximal LL (L1-L4), distal LL (L4-S1), total LL (L1-S1), segmental lordosis, pelvic incidence-LL mismatch, thoracic kyphosis, cervical lordosis, and sagittal vertical axis were measured on preoperative and postoperative radiographs. RESULTS: Forty-eight patients were identified. Immediate postoperative radiographs were obtained at a mean (SD) of 17 (20) days after surgery; delayed radiographs were obtained 184 (82) days after surgery. After surgery, patients had significantly decreased pelvic tilt (15.71° [7.25°] vs. 17.52° [7.67°], P = 0.003) and proximal LL (11.86° [10.67°] vs. 16.03° [10.45°], P < 0.001) and increased sacral slope (39.49° [9.27°] vs. 36.31° [10.39°], P < 0.001), LL (55.35° [13.15°] vs. 51.63° [13.38°], P = 0.001), and distal LL (43.17° [9.33°] vs. 35.80° [8.02°], P < 0.001). Segmental lordosis increased significantly at L5-S1 and decreased significantly at L2-3, L3-4, and L4-5. Lordosis distribution index increased from 72.55 (19.53) to 81.38 (22.83) (P < 0.001). CONCLUSIONS: L5-S1 ALIF was associated with increased L5-S1 segmental lordosis accompanied by pelvic anteversion and a reciprocal decrease in proximal LL. These changes may represent a reversal of compensatory mechanisms, suggesting an overall relaxation of spinopelvic alignment after L5-S1 ALIF.

From periphery to center stage: 50 years of advancements in innate immunity.

Over the past 50 years in the field of immunology, something of a Copernican revolution has happened. For a long time, immunologists were mainly concerned with what is termed adaptive immunity, which involves the exquisitely specific activities of lymphocytes. But the other arm of immunity, so-called "innate immunity," had been neglected. To celebrate Cell's 50th anniversary, we have put together a review of the processes and components of innate immunity and trace the seminal contributions leading to the modern state of this field. Innate immunity has joined adaptive immunity in the center of interest for all those who study the body's defenses, as well as homeostasis and pathology. We are now entering the era where therapeutic targeting of innate immune receptors and downstream signals hold substantial promise for infectious and inflammatory diseases and cancer.

Unlocking potential: the role of the electron transport chain in immunometabolism.

The electron transport chain (ETC) couples electron transfer with proton pumping to generate ATP and it also regulates particular innate and adaptive immune cell function. While NLRP3 inflammasome activation was initially linked to reactive oxygen species (ROS) produced from Complexes I and III, recent research suggests that an intact ETC fueling ATP is needed. Complex II may be responsible for Th1 cell proliferation and in some cases, effector cytokine production. Complex III is required for regulatory T (Treg) cell function, while oxidative phosphorylation (OXPHOS) and Complexes I, IV, and V sustain proliferation and antibody production in B lymphocytes, with OXPHOS also being required for B regulatory (Breg) cell function. Despite challenges, the ETC shows therapeutic targeting potential for immune-related diseases and in immuno-oncology.

Itaconate boosts malaria via induction of PD-L1.

The Krebs-cycle-derived metabolite itaconate has been shown to be immunomodulatory, targeting multiple processes in macrophages. Ramalho et al. reveal an additional role for itaconate in malaria.1Plasmodium Chabaudi induces itaconate in dendritic cells (DCs), leading to programmed death-ligand 1 (PD-L1) induction. This suppresses CD8+ T cells, important for host defense against malaria, thereby promoting parasitemia.

Metabolic regulation of type I interferon production.

Over the past decade, there has been a surge in discoveries of how metabolic pathways regulate immune cell function in health and disease, establishing the field of immunometabolism. Specifically, pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, and those involving lipid metabolism have been implicated in regulating immune cell function. Viral infections cause immunometabolic changes which lead to antiviral immunity, but little is known about how metabolic changes regulate interferon responses. Interferons are critical cytokines in host defense, rapidly induced upon pathogen recognition, but are also involved in autoimmune diseases. This review summarizes how metabolic change impacts interferon production. We describe how glycolysis, lipid metabolism (specifically involving eicosanoids and cholesterol), and the TCA cycle-linked intermediates itaconate and fumarate impact type I interferons. Targeting these metabolic changes presents new therapeutic possibilities to modulate type I interferons during host defense or autoimmune disorders.

A break in mitochondrial endosymbiosis as a basis for inflammatory diseases.

Mitochondria retain bacterial traits due to their endosymbiotic origin, but host cells do not recognize them as foreign because the organelles are sequestered. However, the regulated release of mitochondrial factors into the cytosol can trigger cell death, innate immunity and inflammation. This selective breakdown in the 2-billion-year-old endosymbiotic relationship enables mitochondria to act as intracellular signalling hubs. Mitochondrial signals include proteins, nucleic acids, phospholipids, metabolites and reactive oxygen species, which have many modes of release from mitochondria, and of decoding in the cytosol and nucleus. Because these mitochondrial signals probably contribute to the homeostatic role of inflammation, dysregulation of these processes may lead to autoimmune and inflammatory diseases. A potential reason for the increased incidence of these diseases may be changes in mitochondrial function and signalling in response to such recent phenomena as obesity, dietary changes and other environmental factors. Focusing on the mixed heritage of mitochondria therefore leads to predictions for future insights, research paths and therapeutic opportunities. Thus, whereas mitochondria can be considered 'the enemy within' the cell, evolution has used this strained relationship in intriguing ways, with increasing evidence pointing to the recent failure of endosymbiosis being critical for the pathogenesis of inflammatory diseases.

4-Octyl Itaconate Alleviates Airway Eosinophilic Inflammation by Suppressing Chemokines and Eosinophil Development.

4-Octyl itaconate (4-OI) is a derivative of the Krebs cycle-derived metabolite itaconate and displays an array of antimicrobial and anti-inflammatory properties through modifying cysteine residues within protein targets. We have found that 4-OI significantly reduces the production of eosinophil-targeted chemokines in a variety of cell types, including M1 and M2 macrophages, Th2 cells, and A549 respiratory epithelial cells. Notably, the suppression of these chemokines in M1 macrophages was found to be NRF2-dependent. In addition, 4-OI can interfere with IL-5 signaling and directly affect eosinophil differentiation. In a model of eosinophilic airway inflammation in BALB/c mice, 4-OI alleviated airway resistance and reduced eosinophil recruitment to the lungs. Our findings suggest that itaconate derivatives could be promising therapeutic agents for the treatment of eosinophilic asthma.

C5aR2 Regulates STING-Mediated Interferon Beta Production in Human Macrophages.

The complement system mediates diverse regulatory immunological functions. C5aR2, an enigmatic receptor for anaphylatoxin C5a, has been shown to modulate PRR-dependent pro-inflammatory cytokine secretion in human macrophages. However, the specific downstream targets and underlying molecular mechanisms are less clear. In this study, CRISPR-Cas9 was used to generate macrophage models lacking C5aR2, which were used to probe the role of C5aR2 in the context of PRR stimulation. cGAS and STING-induced IFN-ß secretion was significantly increased in C5aR2 KO THP-1 cells and C5aR2-edited primary human monocyte-derived macrophages, and STING and IRF3 expression were increased, albeit not significantly, in C5aR2 KO cell lines implicating C5aR2 as a regulator of the IFN-ß response to cGAS-STING pathway activation. Transcriptomic analysis by RNAseq revealed that nucleic acid sensing and antiviral signalling pathways were significantly up-regulated in C5aR2 KO THP-1 cells. Altogether, these data suggest a link between C5aR2 and nucleic acid sensing in human macrophages. With further characterisation, this relationship may yield therapeutic options in interferon-related pathologies.

Characterization and inhibition of inflammasome responses in severe and non-severe asthma.

BACKGROUND: Increased airway NLRP3 inflammasome-mediated IL-1ß responses may underpin severe neutrophilic asthma. However, whether increased inflammasome activation is unique to severe asthma, is a common feature of immune cells in all inflammatory types of severe asthma, and whether inflammasome activation can be therapeutically targeted in patients, remains unknown. OBJECTIVE: To investigate the activation and inhibition of inflammasome-mediated IL-1ß responses in immune cells from patients with asthma. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with non-severe (n = 59) and severe (n = 36 stable, n = 17 exacerbating) asthma and healthy subjects (n = 39). PBMCs were stimulated with nigericin or lipopolysaccharide (LPS) alone, or in combination (LPS + nigericin), with or without the NLRP3 inhibitor MCC950, and the effects on IL-1ß release were assessed. RESULTS: PBMCs from patients with non-severe or severe asthma produced more IL-1ß in response to nigericin than those from healthy subjects. PBMCs from patients with severe asthma released more IL-1ß in response to LPS + nigericin than those from non-severe asthma. Inflammasome-induced IL-1ß release from PBMCs from patients with severe asthma was not increased during exacerbation compared to when stable. Inflammasome-induced IL-1ß release was not different between male and female, or obese and non-obese patients and correlated with eosinophil and neutrophil numbers in the airways. MCC950 effectively suppressed LPS-, nigericin-, and LPS + nigericin-induced IL-1ß release from PBMCs from all groups. CONCLUSION: An increased ability for inflammasome priming and/or activation is a common feature of systemic immune cells in both severe and non-severe asthma, highlighting inflammasome inhibition as a universal therapy for different subtypes of disease.

Fumarate hydratase as a metabolic regulator of immunity.

Tricarboxylic acid (TCA) cycle metabolites have been implicated in modulating signalling pathways in immune cells. Notable examples include succinate and itaconate, which have pro- and anti-inflammatory roles, respectively. Recently, fumarate has emerged as having specific roles in macrophage activation, regulating the production of such cytokines as interleukin (IL)-10 and type I interferons (IFNs). Fumarate hydratase (FH) has been identified as a control point. Notably, FH loss in different models and cell types has been found to lead to DNA and RNA release from mitochondria which are sensed by cytosolic nucleic acid sensors including retinoic acid-inducible gene (RIG)-I, melanoma differentiation-associated protein (MDA)5, and cyclic GMP-AMP synthase (cGAS) to upregulate IFN-ß production. These findings may have relevance in the pathogenesis and treatment of diseases associated with decreased FH levels such as systemic lupus erythematosus (SLE) or FH-deficient kidney cancer.

Stable Regional and Global Alignment in Patients Treated With Minimally Invasive Lateral Retropleural Thoracic Diskectomy Without Fixation.

BACKGROUND AND OBJECTIVES: Thoracic disk herniations are challenging to treat, and open transthoracic or minimally invasive thoracoscopic approaches are associated with significant morbidity, substantial costs, and steep learning curves. The minimally invasive lateral retropleural thoracic diskectomy (MIS-LRP-TD) approach is straightforward and is associated with lower perioperative morbidity. With MIS-LRP-TD, the overlying rib, ipsilateral pedicle, ligamentum flavum, posterior longitudinal ligament, and posterior third of the adjacent vertebral bodies are resected. Adjunct fixation is typically not performed, eliminating hardware-related complications and costs. This radiographic study investigates long-term global and thoracic spine alignment after MIS-LRP-TD without fixation. METHODS: This study was a single-institution, retrospective evaluation of all patients who underwent MIS-LRP-TD without fixation between November 7, 2017 and July 19, 2022. Preoperative and the most recent postoperative radiographs were used to determine the C7 plumb line to central sacral vertical line, thoracic Cobb angle (TCA), segmental Cobb angle, C7 to sagittal vertical axis, thoracic kyphosis, and segmental kyphosis. RESULTS: In total, 22 patients with 24 disk herniations underwent MIS-LRP-TD without fixation. The mean (SD) radiographic follow-up was 12.9 (11.2) months. Overall, no significant differences were seen in C7 plumb line to central sacral vertical line (P = .65), C7 to sagittal vertical axis (P = .99), thoracic kyphosis (P = .30), TCA (P = .28), segmental kyphosis (P = .27), or segmental Cobb angle (P = .56) at follow-up. One patient demonstrated a >5° change in TCA but remained asymptomatic. CONCLUSION: Despite requiring extensive resection of the middle column and ipsilateral costovertebral joint at the index level, MIS-LRP-TD without adjunct fixation does not lead to significant global, regional, or segmental deformity. Thus, MIS-LRP-TD appears to be a safe, effective treatment approach for challenging thoracic disk herniations.

4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1.

IMPORTANCE: Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics.

A comparison of modern-era anterior lumbar interbody fusion and minimally invasive transforaminal lumbar interbody fusion at the lumbosacral junction.

OBJECTIVE: Interbody fusion is the primary method for achieving arthrodesis across the lumbosacral junction in the setting of degenerative pathologies, such as spondylosis and spondylolisthesis. Two common techniques are anterior lumbar interbody fusion (ALIF) and posterior transforaminal lumbar interbody fusion (TLIF). In recent years, interbody design and technology have advanced, and most earlier studies comparing ALIF and TLIF did not specifically assess the lumbosacral junction. This study compared changes in radiographic and clinical parameters between patients undergoing modern-era single-level ALIF and minimally invasive surgery (MIS) TLIF at L5-S1. METHODS: Consecutive patients who underwent single-segment L5-S1 ALIF or MIS TLIF performed by the senior authors over a 6-year interval (January 1, 2016-November 30, 2021) were retrospectively reviewed. Upright radiographs were used to determine pre- and postoperative lumbar lordosis, segmental lordosis, disc angle, and neuroforaminal height. Improvements in patient-reported outcome scores (Oswestry Disability Index and SF-36) were also compared. RESULTS: Overall, 108 patients (58 [54%] men, 50 [46%] women; mean [SD] age 57.6 [13.5] years) were included in the study. ALIF was performed in 49 patients, and TLIF was performed in 59 patients. The most common treatment indications were spondylolisthesis (50%, 54/108) and spondylosis (46%, 50/108). The cohorts did not differ in terms of intraoperative (p > 0.99) or postoperative (p = 0.73) complication rates. The mean (SD) hospital length of stay was significantly shorter for patients undergoing TLIF than ALIF (1.3 [0.6] days vs 2.0 [1.4] days, p < 0.001). Both techniques significantly improved L5-S1 segmental lordosis, disc angle, and neuroforaminal height (p ≤ 0.008). ALIF versus TLIF significantly increased mean [SD] segmental lordosis (12.5° [7.3°] vs 2.0° [5.7°], p < 0.001), disc angle (14.8° [5.5°] vs 3.0° [6.1°], p < 0.001), and neuroforaminal height (4.5 [4.6] mm vs 2.4 [3.0] mm, p = 0.008). Improvements in patient-reported outcome parameters and reoperation rates were similar between cohorts. CONCLUSIONS: When treating patients at a single segment across the lumbosacral junction, ALIF resulted in significantly greater increases in segmental lordosis, L5-S1 disc angle, and neuroforaminal height compared with MIS TLIF. Improvements in clinical parameters and reoperation rates were similar between the 2 techniques.

4-Octyl Itaconate and Dimethyl Fumarate Induce Secretion of the Anti-Inflammatory Protein Annexin A1 via NRF2.

Annexin A1 is a key anti-inflammatory effector protein that is involved in the anti-inflammatory effects of glucocorticoids. 4-Octyl itaconate (4-OI), a derivative of the endogenous metabolite itaconate, which is abundantly produced by LPS-activated macrophages, has recently been identified as a potent anti-inflammatory agent. The anti-inflammatory effects of 4-OI share a significant overlap with those of dimethyl fumarate (DMF), a derivate of another Krebs cycle metabolite fumarate, which is already in use clinically for the treatment of inflammatory diseases. In this study we show that both 4-OI and DMF induce secretion of the 33-kDa form of annexin A1 from murine bone marrow-derived macrophages, an effect that is much more pronounced in LPS-stimulated cells. We also show that this 4-OI- and DMF-driven annexin A1 secretion is NRF2-dependent and that other means of activating NRF2 give rise to the same response. Lastly, we demonstrate that the cholesterol transporter ABCA1, which has previously been implicated in annexin A1 secretion, is required for this process in macrophages. Our findings contribute to the growing body of knowledge on the anti-inflammatory effects of the Krebs cycle metabolite derivatives 4-OI and DMF.

NLRP12 drives PANoptosis in response to heme.

PANoptosomes are large cell death-inducing complexes that drive a type of cell death called PANoptosis during infection and inflammation. Sundaram and colleagues recently identified NLRP12 as a PANoptosome that induces PANoptosis in response to heme, TNF, and pathogen-associated molecular patterns (PAMPs), indicating a role for NLRP12 in hemolytic and inflammatory diseases.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.