Autoregulation of insulin receptor signaling through MFGE8 and the αvβ5 integrin

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvβ5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/β5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvβ5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/β5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvβ5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

Acute insulin resistance can be viewed as a protective response under specific physiological conditions that necessitate increased insulin secretion. Nevertheless, the increasing prevalence of chronic insulin resistance (1) in the current obesity epidemic hastens the development of type 2 diabetes (T2D) and induces compensatory hyperinsulinemia. Hyperinsulinemia can produce potentially maladaptive consequences at least in part, due to the mitogenic roles of insulin (2–4). As such, there remains a critical need for new therapies to improve insulin sensitivity in order to prevent T2D, avoid the need for insulin treatment in patients with T2D, or reduce the insulin dose required to normalize blood glucose in such individuals.Insulin binding to the alpha subunit of the insulin receptor induces a conformational change that triggers activation of insulin receptor beta subunit (IRβ) tyrosine kinase activity (5–7). The activated insulin receptor phosphorylates target molecules that mediate downstream signaling leading to glucose uptake and other metabolic effects (8, 9). Dephosphorylation of IRβ and insulin receptor substrate-1 (IRS-1) aids in termination of insulin signaling pathways (10, 11) and is the basis of clinical trials targeting putative phosphatases to treat diabetes (12). Despite their potential therapeutic relevance, there is a relative paucity of knowledge regarding molecular mechanisms that lead to termination of insulin receptor signaling.The integrin families of cell surface receptors mediate bidirectional signaling between the cell and its external environment. Previous work has identified interactions between integrin receptors and other growth factor receptor tyrosine kinases (13–16) that lead to modulation of downstream signaling (17–19). For example, the αvβ3 and α6β4 integrins function as coreceptors for insulin-like growth factor-1 and 2 (IGF1 and 2) and potentiate IGF1 receptor (IGF1R)-mediated signaling (19–23). Immunoprecipitation studies have demonstrated a physical association between the αv integrins and IRβ (24, 25). The impact of these associations on glucose homeostasis has not been evaluated. A role for β1 integrins in the regulation of glucose homeostasis is well established. This class of integrins appears to be particularly important in regulating insulin-mediated glucose homeostasis in the obese state. The effect of β1 integrins on glucose homeostasis appears to be primarily due to obesity-associated matrix remodeling (26–30) rather than a direct effect secondary to a physical association between β1 integrins and the insulin receptor.Milk fat globule epidermal growth factor like 8 (MFGE8) is a secreted integrin ligand which binds the αvβ3, αvβ5, and α8β1 integrins (31, 32). Several recent observations suggest a role for MFGE8 in modulating insulin resistance. In humans, serum MFGE8 levels are increased in the context of diabetes and correlate positively with the extent of hemoglobin glycosylation (33, 34). Indeed, serum MFGE8 levels correlate with indices of insulin resistance in two independent cohorts of patients with T2D or gestational diabetes from China (35, 36). A missense variation in the gene encoding MFGE8, present in South Asian Punjabi Sikhs, is associated with increased circulating MFGE8 levels and increased risk of developing T2D (37). Increased circulating levels of MFGE8 in diabetic patients may impact T2D through effects on inflammation and cardiovascular disease. Humans with increased MFGE8 expression have a greater risk of developing coronary artery disease (38). In contrast, in murine models, MFGE8 deficiency exacerbates cardiac hypertrophy and atherosclerosis (39, 40). MFGE8 also improves wound healing responses in diabetic foot ulcers (41, 42) by triggering apoptotic cell clearance and promoting resolution of inflammation (43–45).Despite the notable links between MFGE8, insulin resistance, and T2D pathology, the biology underlying these associations has not been investigated. We therefore evaluated the effect of acute antibody-mediated disruption of the MFGE8/β5 pathway on glucose homeostasis in wild-type (WT) mice. We report here that MFGE8 markedly attenuates the effect of insulin on skeletal muscle glucose uptake. Antibody-mediated blockade of MFGE8 or αvβ5 enhances while recombinant MFGE8 (rMFGE8) reduces insulin-stimulated glucose uptake in vitro and in vivo. Mechanistically, insulin acts to promotes cell-surface enrichment of skeletal muscle MFGE8, which then binds to cell surface αvβ5 and increases the interaction between the integrin and the insulin receptor. This interaction subsequently aids in terminating insulin receptor signaling.     

Cytocidal macrophages in symbiosis with CD4 and CD8 T cells cause acute diabetes following checkpoint blockade of PD-1 in NOD mice

Autoimmune diabetes is one of the complications resulting from checkpoint blockade immunotherapy in cancer patients, yet the underlying mechanisms for such an adverse effect are not well understood. Leveraging the diabetes-susceptible nonobese diabetic (NOD) mouse model, we phenocopy the diabetes progression induced by programmed death 1 (PD-1)/PD-L1 blockade and identify a cascade of highly interdependent cellular interactions involving diabetogenic CD4 and CD8 T cells and macrophages. We demonstrate that exhausted CD8 T cells are the major cells that respond to PD-1 blockade producing high levels of IFN-γ. Most importantly, the activated T cells lead to the recruitment of monocyte-derived macrophages that become highly activated when responding to IFN-γ. These macrophages acquire cytocidal activity against β-cells via nitric oxide and induce autoimmune diabetes. Collectively, the data in this study reveal a critical role of macrophages in the PD-1 blockade-induced diabetogenesis, providing new insights for the understanding of checkpoint blockade immunotherapy in cancer and infectious diseases.

Two of the most widely studied proteins that regulate T cell activation are cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1). Inhibiting them can unleash regulatory controls of T cell activation, allowing the T cells to display their full functional potential. CTLA-4 and PD-1 are extensively studied in the cancer field, where their inhibitors—monoclonal antibodies—are used to treat cancer patients. Despite achieving success clinically, the checkpoint blockade immunotherapy can result in immune-related adverse events that frequently include endocrine autoimmune diseases (1), among them type 1 diabetes (T1D) (2–4). A recent comprehensive review summarized the clinical features of T1D induced by the immune checkpoint inhibition (4).To better understand how PD-1 is regulating T1D, we examine here the nonobese diabetic (NOD) mouse model for changes in various cellular components following PD-1 blockade. PD-1 is synthesized de novo in activated T cells mediated by T cell receptor (TCR) signaling (5–7). Whereas PD-1 expression is rapidly up-regulated after antigen stimulation of naïve T cells, sustained TCR stimulation results in substantially higher expression of PD-1 and the establishment of T cell exhaustion in the examples of chronic viral infection and cancers (8–13). NOD mice with a gene knockout of PD-1 or treated with PD-1 blocking antibody develop accelerated autoimmune diabetes shown in a number of studies (14–21). Both autoreactive CD4 and CD8 T cells can respond to PD-1/PD-L1 blockade and contribute to the acute diabetes development (17–19). Central in this process is PD-L1 (the ligand of PD-1) expressed by the islet parenchymal cells (20, 21), which limits the T cell function in the islets and protects against autoimmune diabetes.In this report we examine changes in various cellular components following PD-1 blockade by single-cell RNA sequencing (scRNA-seq) and identify a previously unexplored islet macrophage population derived from monocytes with high proinflammatory activity. In the islets of anti–PD-1–treated mice, the infiltration by monocyte-derived macrophage (MoMac) was under the influence of CD4 and particularly CD8 T cells. The CD8 T cells largely comprised the precursor exhausted T (T_PEX) cells that were activated and differentiated to produce abundant IFN-γ in response to PD-1 blockade, which in turn activated the infiltrated MoMac to promote diabetes progression. The anti–PD-1 induced development of acute diabetes was reduced by restricting the infiltration and function of such MoMac in islets. Our study establishes that the myeloid cell compartment is an indispensable component of PD-1 regulation in autoimmune diabetes. Our study provides a cellular target, the MoMac, that may minimize the adverse effects of checkpoint blockade immunotherapy.     

CCRL2 promotes antitumor T-cell immunity via amplifying TLR4-mediated immunostimulatory macrophage activation

Macrophages are the key regulator of T-cell responses depending on their activation state. C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical receptor originally cloned from LPS-activated macrophages, has recently been shown to regulate immune responses under several inflammatory conditions. However, whether CCRL2 influences macrophage function and regulates tumor immunity remains unknown. Here, we found that tumoral CCRL2 expression is a predictive indicator of robust antitumor T-cell responses in human cancers. CCRL2 is selectively expressed in tumor-associated macrophages (TAM) with immunostimulatory phenotype in humans and mice. Conditioned media from tumor cells could induce CCRL2 expression in macrophages primarily via TLR4, which is negated by immunosuppressive factors. Ccrl2^−/− mice exhibit accelerated melanoma growth and impaired antitumor immunity characterized by significant reductions in immunostimulatory macrophages and T-cell responses in tumor. Depletion of CD8⁺ T cells or macrophages eliminates the difference in tumor growth between WT and Ccrl2^−/− mice. Moreover, CCRL2 deficiency impairs immunogenic activation of macrophages, resulting in attenuated antitumor T-cell responses and aggravated tumor growth in a coinjection tumor model. Mechanically, CCRL2 interacts with TLR4 on the cell surface to retain membrane TLR4 expression and further enhance its downstream Myd88-NF-κB inflammatory signaling in macrophages. Similarly, Tlr4^−/− mice exhibit reduced CCRL2 expression in TAM and accelerated melanoma growth. Collectively, our study reveals a functional role of CCRL2 in activating immunostimulatory macrophages, thereby potentiating antitumor T-cell response and tumor rejection, and suggests CCLR2 as a potential biomarker candidate and therapeutic target for cancer immunotherapy.

The central role of T cells, particularly cytotoxic CD8⁺ T cells (CTL), in anti-tumor immunity has been highlighted by the clinical success of cancer immunotherapies. Melanoma is known as an immunogenic tumor with abundant tumor-infiltrating T cells and is susceptible to immune checkpoint blockades (1). However, many types of cancer are not responsive to immunotherapy, and even for melanoma, less than 40% of patients could benefit from these therapies, possibly due to insufficient activation of tumor-specific CTL or their failure to infiltrate tumors (2).Macrophages constitute the largest fraction of tumor-infiltrating immune cells and act as an important regulator during cancer progression (3–6). The abundance of tumor-associated macrophages (TAM) is generally associated with impaired anti-tumor T-cell immunity and poor clinical outcome and response to treatment in solid tumors (7–10). However, in some cases, macrophages can be associated with a good prognosis; for example, high frequencies of HLA-DR⁺ macrophages within tumors have been associated with good outcomes (11–13). It has become clear that TAM consist of a continuum of phenotypes, ranging from an immunostimulatory M1-like phenotype to an immunosuppressive M2-like phenotype (14, 15). M1-like macrophages predominate at sites of early oncogenesis, mediating anti-tumor effects including direct killing and activation of anti-tumor T-cell immunity (5, 7, 16–18). Over tumor progression, macrophages can be shifted toward M2-like phenotype by responding to cues within the tumor microenvironment (TME) (19–21). M2-like macrophages predominate in established tumors, mediating protumor effects including the induction of immunosuppression, promotion of angiogenesis, and tumor cell biology (5, 7). Thus, targeting macrophages has become an attracting strategy to complement the existing cancer immunotherapy. Instead of depletion of all macrophages which contain both anti- and protumor subsets, induction of immunostimulatory phenotype or reprograming TAM from protumor into anti-tumor phenotype could be more efficient to control tumor progression primarily by enhancing anti-tumor T-cell responses (7). Thus, identification of the key factors that regulate the activation state of macrophages, particularly those enforcing anti-tumor M1-like phenotype, could facilitate the development of new therapeutic targets to improve the efficacy of anti-cancer immunotherapy.C-C motif chemokine receptor-like 2 (CCRL2) was originally cloned from LPS-stimulated macrophages and first named as a LPS inducible C-C chemokine receptor related gene (l-CCR) (22). CCRL2 is absent in resting immune cells and induced in activated myeloid cells, but not T cells, under certain pathological conditions (23–27). CCRL2 was later identified as a nonsignaling atypical receptor to enrich and present its ligand chemerin to the functional receptor, CMKLR1 (24). Further studies demonstrated that CCRL2 expressed in endothelial cells promotes CMKLR1-dependent dendritic cell (DC) and natural killer (NK) cell transmigration (28, 29). In addition, CCRL2 expression in activated neutrophils regulates CXCR2-dependent neutrophil chemotaxis toward CXCL8 (25). Surprisingly, the role of CCRL2 in macrophages remains unknown. Preclinical mouse studies demonstrated that CCRL2 is involved in several inflammatory diseases (25, 27, 30). However, the involvement of CCRL2 in tumors has been reported until very recently. CCRL2 expression in nonhematopoietic cells inhibits lung tumors by facilitating NK cell migration (29), while CCRL2 expression in human breast cancer tissues positively correlates to tumor-infiltrating immune cells (31).Here, we demonstrate that CCLR2 expression is not only a predictive indicator of robust anti-tumor immunity in human cancers but also plays a functional role in the activation of immunostimulatory macrophages via interacting with surface TLR4 and amplifying its downstream inflammatory signaling, finally leading to optimal anti-tumor T-cell responses.     

Retinol binding protein 4 primes the NLRP3 inflammasome by signaling through Toll-like receptors 2 and 4

Adipose tissue (AT) inflammation contributes to systemic insulin resistance. In obesity and type 2 diabetes (T2D), retinol binding protein 4 (RBP4), the major retinol carrier in serum, is elevated in AT and has proinflammatory effects which are mediated partially through Toll-like receptor 4 (TLR4). We now show that RBP4 primes the NLRP3 inflammasome for interleukin-1β (IL1β) release, in a glucose-dependent manner, through the TLR4/MD2 receptor complex and TLR2. This impairs insulin signaling in adipocytes. IL1β is elevated in perigonadal white AT (PGWAT) of chow-fed RBP4-overexpressing mice and in serum and PGWAT of high-fat diet-fed RBP4-overexpressing mice vs. wild-type mice. Holo- or apo-RBP4 injection in wild-type mice causes insulin resistance and elevates PGWAT inflammatory markers, including IL1β. TLR4 inhibition in RBP4-overexpressing mice reduces PGWAT inflammation, including IL1β levels and improves insulin sensitivity. Thus, the proinflammatory effects of RBP4 require NLRP3-inflammasome priming. These studies may provide approaches to reduce AT inflammation and insulin resistance in obesity and diabetes.

The incidence of type 2 diabetes (T2D) continues to increase worldwide and constitutes a major global health threat (1). Insulin resistance in muscle, adipose tissue (AT), and liver is a hallmark of T2D, which is associated with low-grade chronic inflammation, characterized by increased serum cytokine levels and a proinflammatory immune profile in AT (2–4). Pattern recognition receptors including Toll-like receptors (TLRs) sense pathogenic microbial components (pathogen-associated molecular patterns, PAMPs) and also host-derived damage-associated molecular patterns (DAMPs), which results in nonpathogen-initiated inflammation often referred to as “sterile inflammation” (5). In obesity, TLR2 and TLR4 are implicated in inflammation-induced insulin resistance in AT, and genetic deletion of TLR2 or TLR4 in mice results in improved insulin sensitivity (6, 7). In obese, insulin-resistant humans, TLR2 and TLR4 expression and activation are increased in AT compared to healthy controls (8) and some evidence suggests that people taking an antiinflammatory agent (abatacept, also known as CTLA4-Ig) which blocks antigen presentation, may have improved insulin sensitivity (9, 10). TLR2 and TLR4 signal through the adaptor proteins myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adaptor-inducing IFN (TRIF) to activate proinflammatory signaling cascades including nuclear factor kappa B (NFκB) and c-Jun N-terminal kinase (JNK) (5, 11). This results in AT immune cell migration, activation, and cytokine secretion which augments insulin resistance in adipocytes (12). Thus, TLR2 and TLR4 contribute to AT inflammation and insulin resistance and their targeting may provide treatment strategies for T2D.Retinol binding protein 4 (RBP4), the major serum retinol transporter, is secreted by the liver and AT (13, 14). Serum RBP4 levels are elevated in obese, insulin-resistant mice and humans (15, 16). Genetic and pharmacological elevation of RBP4 induces insulin resistance in wild-type (WT) mice (16, 17) and reducing RBP4 levels improves insulin sensitivity (16, 18). In humans, RBP4 levels are elevated with prediabetes and correlate positively with many metabolic syndrome-related components, including increased waist/hip ratio, intraabdominal fat mass, dyslipidemia, hypertension, and cardiovascular disease in large epidemiological studies (15, 19–21). A gain-of-function polymorphism in the RBP4 promoter which increases adipose RBP4 expression is associated with an 80% increased risk of T2D in humans (19, 22).Inflammation is critical for RBP4-induced insulin resistance which is partially mediated through TLR4 (17, 18, 23). RBP4 induces insulin resistance in adipocytes indirectly by increasing proinflammatory cytokine secretion from macrophages (23). Insulin resistance and AT inflammation have been observed in two mouse models of RBP4 overexpression. Mice overexpressing RBP4 driven by a muscle-specific promoter (RBP4-Ox) have elevated serum and perigonadal white adipose tissue (PGWAT) RBP4 protein levels and PGWAT inflammation (17). Elevated AT RBP4 in these mice activates antigen presentation through the JNK pathway which results in proinflammatory CD4 T cell proliferation and Th1 polarization (17). Mice overexpressing RBP4 selectively in adipocytes also have AT inflammation and glucose intolerance even on a chow diet (14). Transfer of RBP4-activated dendritic cells into normal mice is sufficient to cause AT inflammation and insulin resistance (17). Interestingly, overexpression of RBP4 selectively in hepatocytes has been reported not to cause elevated circulating RBP4 levels and is not associated with insulin resistance (24) even though hepatocytes are thought to be the major site for RBP4 secretion (25). Adipocytes can contribute to circulating RBP4 levels especially in obesity (14). Taken together, these data suggest that RBP4 in AT may be an obesity and insulin-resistance-related damage-associated molecule pattern.Activation of PAMP and DAMP receptors in immune cells leads to the assembly of inflammasomes, which are multiprotein complexes that cleave and activate interleukin-1 beta (IL1β) and interleukin-18 (IL18) (26). The nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) inflammasome plays a role in the pathogenesis of obesity, type 1 diabetes, type 2 diabetes, and metabolic syndrome (26–28). NLRP3 knockout (KO) protects against insulin resistance in mice (27). NLRP3 activation requires a two-step process. The first “priming” occurs in response to TLR-mediated activation of the NFκB pathway resulting in expression of pro-IL1β (26). The second “activating” step directly induces inflammasome assembly which recruits and cleaves procaspase-1 to its active form caspase-1 (26). Caspase-1 mediates the cleavage of pro-IL1β resulting in IL1β release (26). The NLRP3 inflammasome can be activated by metabolites which are elevated in obesity and insulin resistance, such as palmitate (29). While an ever-expanding list of several metabolites is known to provide the second signal in inflammasome activation, there is a paucity of data on the endogenous proteins and metabolites that provide the first priming signal in obesity and T2D. Here we show that RBP4 is an endogenous NLRP3-inflammasome priming agent and we investigate its upstream signaling pathways.Our data show that elevating RBP4 levels by RBP4 injection in WT mice or genetically induced RBP4 overexpression markedly elevates adipose IL1β expression, which leads to PGWAT inflammation and insulin resistance. IL1β is elevated in PGWAT of chow-fed RBP4-Ox mice and in serum and PGWAT of HFD-fed RBP4-Ox mice compared to WT mice. The RBP4-mediated proinflammatory effects are mediated specifically through TLR2 and a TLR4/MD2 receptor complex, which does not require the other adaptor proteins LPS-binding protein and CD14. The activation of macrophages by RBP4 through TLR2 and TLR4/MD2 requires signaling through the downstream pathways MyD88 and TRIF. TLR4 inhibition in RBP4-Ox reduces IL1β levels in PGWAT and improves insulin sensitivity. The RBP4-mediated increase in IL1β release from macrophages is glucose dependent. Thus, targeting the NLRP3 inflammasome or the upstream activating receptors or pathways may provide therapeutic avenues to ameliorate RBP4-mediated insulin resistance and T2D.     

DLL1 orchestrates CD8+ T cells to induce long-term vascular normalization and tumor regression

The immunosuppressive and hypoxic tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we showed that elevated levels of Delta-like 1 (DLL1) in the breast and lung TME induced long-term tumor vascular normalization to alleviate tumor hypoxia and promoted the accumulation of interferon γ (IFN-γ)–expressing CD8⁺ T cells and the polarization of M1-like macrophages. Moreover, increased DLL1 levels in the TME sensitized anti-cytotoxic T lymphocyte–associated protein 4 (anti-CTLA4) treatment in its resistant tumors, resulting in tumor regression and prolonged survival. Mechanically, in vivo depletion of CD8⁺ T cells or host IFN-γ deficiency reversed tumor growth inhibition and abrogated DLL1-induced tumor vascular normalization without affecting DLL1-mediated macrophage polarization. Together, these results demonstrate that elevated DLL1 levels in the TME promote durable tumor vascular normalization in a CD8⁺ T cell– and IFN-γ–dependent manner and potentiate anti-CTLA4 therapy. Our findings unveil DLL1 as a potential target to persistently normalize the TME to facilitate cancer immunotherapy.

One of the major challenges currently facing cancer treatments is the aberrant tumor microenvironment (TME), characterized as hypoxia, immunosuppression, acidity, and high interstitial fluid pressure (IFP) (1–5). These properties render tumors resistant to many kinds of cancer treatment modalities. High IFP prevents the penetration and distribution of drug agents into the tumor parenchyma, while hypoxia compromises the effectiveness of chemotherapy and radiotherapy because both treatment modalities often require reactive oxygen species to evoke antitumor activities (4, 6). In addition, hypoxia induces the secretion of multiple immune inhibitory factors and promotes the accumulation of immune regulatory cell populations, such as transforming growth factor-β (TGF-β), interleukin 10 (IL10), myeloid-derived suppressor cells (MDSCs), M2-like tumor-associated macrophages (M2-TAMs), and regulatory T cells (Tregs) (1, 3, 7–9). Thus, the hypoxic and immunosuppressive TME hinders cancer immunotherapy to efficiently eradicate cancer cells.Emerging evidence suggests that the abnormal tumor vasculature contributes largely to the aberrant TME (1, 3, 4). Tumor blood vessels are tortuous, dilated, and leaky with low pericyte coverage. The resulting blood flow is often static and fluctuated and therefore creates a hypoxic and acidic TME with high IFP (4). Therefore, tumor vascular normalization has been proposed as a promising approach to alleviate the aberrances within the TME, thus enhancing the efficacy of a range of cancer treatment modalities, including chemotherapy, radiotherapy, and immunotherapy (10–18). Vascular endothelial growth factor (VEGF) ligands and receptors constitute one of the most potent proangiogenic signaling pathways (19). Various VEGF signaling inhibitors, such as Bevacizumab and Cediranib, have been approved to treat several types of cancers. VEGF signaling inhibitors can induce tumor vascular normalization; however, the duration of the normalization is usually transient, and therefore, the improvement to the concurrent chemotherapy and immunotherapy is marginal (4, 19–21). In addition, many kinds of cancer are intrinsically resistant to VEGF signaling targeted therapy (4, 19). Thus, novel approaches are needed to induce tumor vascular normalization for longer periods and in broad tumor types.The evolutionarily conserved Notch signaling pathway plays critical roles in cell differentiation and blood vessel formation. The Notch signaling pathway consists of four Notch receptors (Notch 1 to 4) and four ligands (Jagged1, Jagged2, Delta-like 1 [DLL1], and DLL4) in murine (22). Both Notch receptors and ligands are membrane proteins. DLL1, DLL4, and Jagged1 have been shown to express in endothelial cells and play important roles in vascular development and postnatal vessel formation (23, 24). DLL1 and DLL4 are also associated with tumor angiogenesis (24–26). DLL4 is usually expressed in tumor endothelial cells but rarely in tumor cells (27, 28). Blockade of DLL4 suppresses tumor growth through the induction of nonfunctional tumor vessel formation (24, 25, 29). Thus, activation of DLL4/Notch signaling has the potential to increase tumor vascular maturation. Indeed, higher expression of DLL4 in bladder tumor endothelial cells was correlated with vessel maturation (30). Unfortunately, long-term DLL4 blockade led to vascular neoplasms, and persistent activation of DLL4/Notch signaling promoted T cell acute lymphoblastic leukemia (T-ALL) (31–33).Because of these potential safety concerns of chronic blockade or activation of DLL4/Notch signaling, we proposed instead to remodel tumor vessels via the activation of DLL1/Notch signaling. In contrast to the extensive attention of DLL4 in tumor angiogenesis, the roles of DLL1 in tumor vessel formation is largely unknown. Here, we showed that overexpression of DLL1 in EO771 breast and LAP0297 lung tumor cells not only induced durable tumor vascular normalization but also stimulated CD8⁺ T cell activities. Interestingly, in vivo depletion of CD8⁺ T cells prior to tumor implantation or host IFN-γ deficiency abrogated the effects of DLL1 overexpression on tumor vessels, suggesting that selective activation of DLL1/Notch signaling induces long-term tumor vascular normalization via T cell activation. Moreover, DLL1/Notch signaling activation in combination with anti-CTLA4 therapy prolonged survival. Thus, this study uncovered DLL1 as a potential target to induce long-term tumor vascular normalization to enhance cancer immunotherapy.     

Stage-specific overcompensation,the hydra effect,and the failure to eradicate an invasive predator

As biological invasions continue to increase globally, eradication programs have been undertaken at significant cost, often without consideration of relevant ecological theory. Theoretical fisheries models have shown that harvest can actually increase the equilibrium size of a population, and uncontrolled studies and anecdotal reports have documented population increases in response to invasive species removal (akin to fisheries harvest). Both findings may be driven by high levels of juvenile survival associated with low adult abundance, often referred to as overcompensation. Here we show that in a coastal marine ecosystem, an eradication program resulted in stage-specific overcompensation and a 30-fold, single-year increase in the population of an introduced predator. Data collected concurrently from four adjacent regional bays without eradication efforts showed no similar population increase, indicating a local and not a regional increase. Specifically, the eradication program had inadvertently reduced the control of recruitment by adults via cannibalism, thereby facilitating the population explosion. Mesocosm experiments confirmed that adult cannibalism of recruits was size-dependent and could control recruitment. Genomic data show substantial isolation of this population and implicate internal population dynamics for the increase, rather than recruitment from other locations. More broadly, this controlled experimental demonstration of stage-specific overcompensation in an aquatic system provides an important cautionary message for eradication efforts of species with limited connectivity and similar life histories.

Theoretical population models can produce counterintuitive predictions regarding the consequences of harvest or removal of predatory species. These models show that for simple predator-prey systems, there can be positive population responses to predator mortality resulting from harvest for fisheries or population management, which can create an increased equilibrium level of that predator species (1–5). Among these mortality processes is the “hydra effect,” named after the mythical multi-headed serpent that grew two new heads for each one that was removed (6, 7). This counterintuitive outcome can be driven by a density-dependent process known as overcompensation. The hydra effect typically refers to higher equilibrium or time-averaged densities in response to increased mortality, typically involving consumer populations undergoing population cycles. Population increases in response to mortality can be the result of stage-specific overcompensation, which involves an increase in a specific life history stage or a size class following increased mortality. The first analysis of overcompensatory responses to mortality did not depend on stage specificity and was applied initially to fisheries harvests (1). Subsequent models have included stage specificity and have been applied to a broad range of systems in which species have been harvested for consumption or removed for population control of non-native species (4, 5, 8–15).Theory suggests that overcompensation in response to harvest or removal can occur for a variety of reasons, including 1) reduced competition for resources and increased adult reproduction rates, 2) faster rates of juvenile maturation or greater success in reaching the adult stage, and 3) increased juvenile or adult survival rates (1–7). An increase in reproductive output in response to reduced adult density can be the result of a reduction in resource competition (SI Appendix, Fig. S1).While there is substantial evidence that conditions that could produce density-dependent overcompensation occur frequently, evidence for overcompensation in natural populations is rare. For only a few populations do we have the long-term demographic data collected over a sufficiently long duration and for population densities over a wide enough range to detect this effect. Unfortunately, recent reviews of population increases in response to increased mortality do not include field studies with explicit controls for removals (13–17).There are examples of density-dependent overcompensation from field populations (4, 13–15), as well as a larger number of studies from the laboratory and greenhouse typically involving plant and insect populations (18–22). Among the field examples is a population control program for smallmouth bass in a lake in upstate New York, which paradoxically resulted in greater bass abundance, primarily of juveniles, after 7 y of removal efforts (23, 24). Another field study in the United Kingdom showed that perch populations responded similarly when an unidentified pathogen decimated adults (25). Other programs that attempted to remove invasive fishes, including pikeperch in England (26), brook trout in Idaho (27), and Tilapia in Australia (28), showed similar results. However, although many of these examples involved well-executed studies with substantial field data, none had explicit controls for removal, such as comparable populations without harvest (or disease). Thus, despite the support of current theory in these studies, the contribution of external factors to observed population responses to harvest remains uncertain. To date, we are unaware of any experimental studies with comparable controls in a field population that demonstrates overcompensation in a single species (13–15).     

Identifying hydrophobic protein patches to inform protein interaction interfaces

Interactions between proteins lie at the heart of numerous biological processes and are essential for the proper functioning of the cell. Although the importance of hydrophobic residues in driving protein interactions is universally accepted, a characterization of protein hydrophobicity, which informs its interactions, has remained elusive. The challenge lies in capturing the collective response of the protein hydration waters to the nanoscale chemical and topographical protein patterns, which determine protein hydrophobicity. To address this challenge, here, we employ specialized molecular simulations wherein water molecules are systematically displaced from the protein hydration shell; by identifying protein regions that relinquish their waters more readily than others, we are then able to uncover the most hydrophobic protein patches. Surprisingly, such patches contain a large fraction of polar/charged atoms and have chemical compositions that are similar to the more hydrophilic protein patches. Importantly, we also find a striking correspondence between the most hydrophobic protein patches and regions that mediate protein interactions. Our work thus establishes a computational framework for characterizing the emergent hydrophobicity of amphiphilic solutes, such as proteins, which display nanoscale heterogeneity, and for uncovering their interaction interfaces.

Protein–protein interactions play a crucial role in numerous biological processes, ranging from signal transduction and immune response to protein aggregation and phase behavior (1–3). Consequently, being able to understand, predict, and modulate protein interactions has important implications for understanding cellular processes and mitigating the progression of disease (4, 5). A necessary first step toward this ambitious goal is uncovering the interfaces through which proteins interact (6–8). In principle, identifying hydrophobic protein regions, which interact weakly with water, should be a promising strategy for uncovering protein interaction interfaces (9, 10). Indeed, the release of weakly interacting hydration waters from hydrophobic regions can drive protein interactions, as well as other aqueous assemblies (11–13). However, even when the structure of a protein is available at atomistic resolution, it is challenging to identify its hydrophobic patches because they are not uniformly nonpolar, but display variations in polarity and charge at the nanoscale. Moreover, the emergent hydrophobicity of a protein patch stems from the collective response of protein hydration waters to the nanoscale chemical and topographical patterns displayed by the patch (14–20) and cannot be captured by simply counting the number of nonpolar groups in the patch, or even through more involved additive approaches, such as hydropathy scales or surface-area models (21–28).To address this challenge, we build upon seminal theoretical advances and molecular simulation studies, which have shown that near a hydrophobic surface, it is easier to disrupt surface–water interactions and form interfacial cavities (29–34). To uncover protein regions that have the weakest interactions with water, here, we employ specialized molecular simulations, wherein protein–water interactions are disrupted by systematically displacing water molecules from the protein hydration shell (35–37). By identifying the protein patches that nucleate cavities most readily in our simulations, we are then able to uncover the most hydrophobic protein regions. Interestingly, we find that both hydrophobic and hydrophilic protein patches are highly heterogeneous and contain comparable numbers of nonpolar and polar atoms. Our results thus highlight the nontrivial relationship between the chemical composition of protein patches and their emergent hydrophobicity (24–26), and further emphasize the importance of accounting for the collective solvent response in characterizing protein hydrophobicity (16). We then interrogate whether the most hydrophobic protein patches, which nucleate cavities readily, are also likely to mediate protein interactions. Across five proteins that participate in either homodimer or heterodimer formation, we find that roughly 60 to 70% of interfacial contacts and only about 10 to 20% of noncontacts nucleate cavities. Our work thus provides a versatile computational framework for characterizing hydrophobicity and uncovering interaction interfaces of not just proteins, but also of other complex amphiphilic solutes, such as cavitands, dendrimers, and patchy nanoparticles (38–41).     

Neural stem cells secreting bispecific T cell engager to induce selective antiglioma activity

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. No treatment provides durable relief for the vast majority of GBM patients. In this study, we''ve tested a bispecific antibody comprised of single-chain variable fragments (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this bispecific T cell engager (BiTE) (BiTE^LLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patients'' tumors and, in so doing, exerts anti-GBM activity ex vivo. The interaction of BiTE^LLON with T cells and IL13Rα2-expressing GBM cells stimulates T cell proliferation and the production of proinflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα). We have modified neural stem cells (NSCs) to produce and secrete the BiTE^LLON (NSC^LLON). When injected intracranially in mice with a brain tumor, NSC^LLON show tropism for tumor, secrete BiTE^LLON, and remain viable for over 7 d. When injected directly into the tumor, NSC^LLON provide a significant survival benefit to mice bearing various IL13Rα2⁺ GBMs. Our results support further investigation and development of this therapeutic for clinical translation.

Routine treatment of newly diagnosed glioblastoma (GBM) consists of surgical resection, chemotherapy, and radiation, which results in a median GBM patient survival of less than 2 y, with just 5% of patients surviving beyond 5 y (1). The blood–brain barrier (BBB) limits therapeutic access to the tumor (2). An immunosuppressive microenvironment and molecular heterogeneity of GBM present a unique set of challenges for developing effective therapies for this type of brain tumor (3–10).The development of treatments for lessening the immunosuppressive effects of GBM represents an active area of preclinical and clinical neuro-oncology research. Many, if not all, approaches being tested involve increasing T cell cytotoxic antitumor activity (11–17). Large numbers of functional cytotoxic tumor-infiltrating lymphocytes (TILs) correlate with improved progression-free survival for GBM patients (18–21). However, the immunosuppressive milieu of GBM impairs T cell cytolytic function, altering the effectiveness of T cell-based therapies for treating GBM (22–26). Numerous lymphocyte-directed treatments are being investigated, including the use of bispecific T cell engagers (BiTEs) (17, 27, 28). BiTEs can be produced and used without patient-specific BiTE individualization and can, therefore, be considered “off-the-shelf” therapeutics (29–32). The use of BiTEs targeting tumor-associated antigens (TAAs) has been approved by the Food and Drug Administration (FDA) in treating liquid malignancies, and BiTE-associated treatments are currently being evaluated in multiple clinical studies for solid tumors (e.g., {"type":"clinical-trial","attrs":{"text":"NCT03792841","term_id":"NCT03792841"}}NCT03792841, {"type":"clinical-trial","attrs":{"text":"NCT04117958","term_id":"NCT04117958"}}NCT04117958, and {"type":"clinical-trial","attrs":{"text":"NCT03319940","term_id":"NCT03319940"}}NCT03319940) (33–36).BiTEs consist of two single-chain variable fragments (scFvs) connected by a flexible linker (37). One of the scFvs is directed to a TAA and the other to CD3 epsilon (ε) that is expressed on T cells (38). BiTEs engage TILs and cancer cells in a major histocompatibility complex (MHC)-independent manner and are, therefore, unaffected by MHC down-regulation that occurs in GBM cells (37–40). The specificity of BiTE''s tumor antigen-directed scFv is imperative to harness the full therapeutic potential of the recombinant molecule (41). BiTE anticancer activity requires BiTE binding with malignant and immune cells simultaneously; single-arm binding to a tumor antigen or CD3ε is therapeutically ineffective (42, 43).The short half-life of BiTEs in plasma necessitates a frequent or continuous infusion into patient circulation to maintain therapeutic levels of BiTE (43–45). Several approaches have been proposed and tested to compensate for the rate of BiTE biologic life in treating peripheral cancers (45–47). These include the recombinant protein''s sustained production by subcutaneous injection of mesenchymal stem cells (MSCs) seeded into a synthetic extracellular matrix scaffold, liver translation of BiTE messenger RNA (mRNA), and peritumoral delivery of MSCs secreting BiTEs. A recent study also explored the use of modified immune cell delivery of BiTE to GBM (48), and the reduction of tumor burden in treated animals has been observed. It remains to be determined if these bicistronic antiglioma treatments share chimeric antigen receptor (CAR) T cells'' fate, which includes a low penetrance and short survival of CARs within the brain (27, 49, 50); both are limiting factors for sustained and efficient delivery of BiTEs by CAR T cells.Neural stem cells (NSCs) have inherent advantages as a cellular carrier of antineoplastic agents to the site of GBM since they are native to the brain. NSCs have demonstrated tropism to brain tumors in several preclinical models. These cells can withstand a harsh oxygen-deprived environment of GBM. Here, we investigated NSCs as producers of BiTEs targeting the tumor-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and their antitumor activity using in vitro and in vivo models of GBM. In vitro, BiTEs show significant antitumor activity when used in cocultures that include T cells harvested from patients’ blood and tumor tissue. In vivo, NSCs modified for BiTE synthesis migrate to a tumor in animal subjects’ brains while functioning as intra- and peritumoral BiTE producers. The following are details of the results from our experiments in characterizing NSCs that produce IL13Rα2-directed BiTEs. We interpret these findings as support for additional safety and efficacy analysis for their potential clinical translation in treating GBM patients.     

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate–BCoA–CPT1A axis for the regulation of immune homeostasis.

Regulatory T (Treg) cells are CD4⁺ T cells expressing Foxp3 that play a key role in immune suppression (1–3). They can be divided into natural Treg (nTreg) and inducible Treg (iTreg) cells (1–3). nTreg cells, which are often referred to as thymic Treg (tTreg), arise during CD4⁺ T cell differentiation in the thymus under the influence of relatively high-avidity interactions of the T cell receptor (TCR) with self-antigens (1–3). iTreg cells, also called peripherally induced Treg (pTreg), develop in secondary lymphoid tissues. In the presence of TGFβ1, naive CD4⁺ T are induced into iTreg cells upon TCR ligation and costimulation by antigen-presenting cells (APCs) in response to non-self antigens, such as allergens, food, and the commensal microbiota (1–3).It has been demonstrated that iTreg cells are enriched in gut-associated lymphoid tissues (GALTs) and are important for the maintenance of intestinal immune homeostasis (3–5). Intestinal iTreg cells were found to be important for the regulation of inflammatory bowel diseases (IBDs), such as Crohn’s disease (CD) and ulcerative colitis (UC), which can potentially affect any portion of the gastrointestinal tract and induce many further complications such as tissue fibrosis, stenosis, fistulas, and colon cancer over time (6). Enhancement of intestinal iTreg function or adoptive transfer of iTreg could significantly alleviate IBDs in mice (7–9).Different types of T cells are featured by distinct metabolic characteristics. Unlike effector CD4⁺ T cells (Teffs), including Th1, Th2, Th9, and Th17 cells, that are mainly reliant on aerobic glycolysis, iTreg cells largely rely on fatty acid oxidation (FAO) (10–12). Accumulating evidence has demonstrated that T cell differentiation is always coupled with metabolic reprogramming (13, 14). For instance, FAO needs to be established in the process of iTreg differentiation. Up-regulation of FAO improved iTreg generation, whereas impairment in FAO compromised iTreg differentiation (12, 13, 15, 16).FAO, comprised of a cyclical series of reactions, demands different fatty acids (FAs), which can be divided into long-, medium-, and short-chain fatty acids (LCFAs, SCFAs, and MCFAs). It dominantly occurs in mitochondria and results in acetyl-CoA (AcCoA), which could be consumed in tricarboxylic acid (TCA) cycle. For the oxidation of LCFA, it initiates from LCFA activation in cytoplasm, resulting in long-chain acyl-CoA. Subsequently, these resulting molecules are converted into long-chain acyl-carnitine by carnitine palmitoyltransferase 1 (CPT1), which is anchored on the mitochondrial outer membrane. Following its shuttling into mitochondria, long-chain acyl-carnitine experiences a chain of reactions to support FAO. Apparently, the transportation of LCFA from cytoplasm into mitochondria is a prerequisite for FAO. CPT1, the rate-limiting enzyme controlling this key step, is thus recognized as a determinant for FAO. In contrast, SCFAs and MCFAs can diffuse across mitochondrial membrane and drive FAO in a CPT1-independent manner (17). Nevertheless, extensive investigations have suggested an important role for CPT1 in iTreg differentiation (12, 13, 15, 16).In recent years, butyrate, a specific type of SCFA produced from fiber-rich diets through microbial fermentation, was shown to play a critical role in the maintenance of intestinal homeostasis and was therefore recognized as an effective ingredient from food (18–24). By modulating distinct types of immune cells, including dendritic cells (18, 19), macrophages (20), and B and T cells (21–24), butyrate contributes to the orchestration of the delicate balance in intestinal immune system. Elegant investigations have elucidated that butyrate is able to facilitate iTreg differentiation by up-regulating Foxp3 expression as a histone deacetylase (HDAC) inhibitor (22, 23). Meanwhile, butyrate, as metabolic fuel and energy source, could also support FAO in colonic epithelial cells (25). However, whether butyrate could regulate FAO to promote iTreg differentiation is unclear.In this study, we found that increased FAO contributed to enhanced iTreg cell differentiation in response to butyrate. Butyrate was processed, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which played a critical role in the control of FAO by targeting CPT1A. We found that BCoA competed with malonyl-CoA (MCoA), the best-known metabolic intermediate inhibiting CPT1A, to unleash CPT1A activity for FAO and thereby iTreg differentiation. Inhibition of ACSS2 to block BCoA generation compromised butyrate-mediated iTreg generation as well as mitigation of mouse colitis. Collectively, we depicted a previously unappreciated mechanism, namely, the butyrate–BCoA–CPT1A regulatory axis, for iTreg differentiation.     

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna–Matthews–Olson (FMO) pigment–protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4–1 and 4–2-1 pathways because the exciton 4–1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4–1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4–2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment–protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

Photosynthetic organisms convert solar photons into chemical energy by taking advantage of the quantum mechanical nature of their molecular systems and the chemistry of their environment (1–4). Antenna complexes, composed of one or more pigment–protein complexes, facilitate the first steps in the photosynthesis process: They absorb photons and determine which proportion of excitations to move to reaction centers, where charge separation occurs (4). In oxic environments, excitations can generate highly reactive singlet oxygen species. These pigment–protein complexes can quench excess excitations in these environments with molecular moieties such as quinones and cysteine residues (1, 5–7).The Fenna–Matthews–Olson (FMO) complex, a trimer of pigment–protein complexes found in the green sulfur bacterium Chlorobaculum tepidum (8), has emerged as a model system to study the photophysical properties of photosynthetic antenna complexes (9–19). Each subunit in the FMO complex contains eight bacteriochlorophyll-a site molecules (Protein Data Bank, ID code: 3ENI) that are coupled to form a basis of eight partially delocalized excited states called excitons (Fig. 1) (20–23). Previous experiments on FMO have observed the presence of long-lived coherences in nonlinear spectroscopic signals at both cryogenic and physiological temperatures (11, 13). The coherent signals are thought to arise from some combination of electronic (24–26), vibrational (16–18), and vibronic (27) coherences in the system (28–30). One previous study reported that the coherent signals in FMO remain unchanged upon mutagenesis of the protein, suggesting that the signals are ground state vibrational coherences (17). Others discuss the role of vibronic coupling, where electronic and nuclear degrees of freedom become coupled (29). Other dimeric model systems have demonstrated the regimes in which these vibronically coupled states produce coherent or incoherent transport and vibronic coherences (31–33). Recent spectroscopic data has suggested that vibronic coupling plays a role in driving efficient energy transfer through photosynthetic complexes (27, 31, 33, 34), but to date there is no direct experimental evidence suggesting that biological systems use vibronic coupling as part of their biological function.Open in a separate windowFig. 1.(Left) Numbered sites and sidechains of cysteines C353 and C49 in the FMO pigment–protein complex (PDB ID code: 3ENI) (20). (Right) Site densities for excitons 4, 2, and 1 in reducing conditions with the energy transfer branching ratios for the WT oxidized and reduced protein. The saturation of pigments in each exciton denotes the relative contribution number to the exciton. The C353 residue is located near excitons 4 and 2, which have most electron density along one side of the complex, and other redox-active residues such as the Trp/Tyr chain. C353 and C49 surround site III, which contains the majority of exciton 1 density. Excitons 2 and 4 are generally delocalized over sites IV, V, and VII.It has been shown that redox conditions affect excited state properties in pigment-protein complexes, yet little is known about the underlying microscopic mechanisms for these effects (1, 9). Many commonly studied light-harvesting complexes—including the FMO complex (20), light-harvesting complex 2 (LH2) (35), the PC645 phycobiliprotein (36), and the cyanobacterial antenna complex isiA (37)—contain redox-active cysteine residues in close proximity to their chromophores. As the natural low light environment of C. tepidum does not necessitate photoprotective responses to light quantity and quality, its primary photoprotective mechanism concerns its response to oxidative stress. C. tepidum is an obligate anaerobe, but the presence of many active anoxygenic genes such as sodB for superoxide dismutase and roo for rubredoxin oxygen oxidoreductase (38) suggests that it is frequently exposed to molecular oxygen (7, 39). Using time-resolved fluorescence measurements, Orf et al. demonstrated that two cysteine residues in the FMO complex, C49 and C353, quench excitons under oxidizing conditions (1), which could protect the excitation from generating reactive oxygen species (7, 40–42). In two-dimensional electronic spectroscopy (2DES) experiments, Allodi et al. showed that redox conditions in both the wild-type and C49A/C353A double-mutant proteins affect the ultrafast dynamics through the FMO complex (9, 43). The recent discovery that many proteins across the evolutionary landscape possess chains of tryptophan and tyrosine residues provides evidence that these redox-active residues may link the internal protein behavior with the chemistry of the surrounding environment (41, 43).In this paper, we present data showing that pigment–protein complexes tune the vibronic coupling of their chromophores and that the absence of this vibronic coupling activates an oxidative photoprotective mechanism. We use 2DES to show that a pair of cysteine residues in FMO, C49 and C353, can steer excitations toward quenching sites in oxic environments. The measured reaction rate constants demonstrate unusual nonmonotonic behavior. We then use a Redfield model to determine how the exciton energy transfer (EET) time constants arise from changing chlorophyll site energies and their system-bath couplings (44, 45). The analysis reveals that the cysteine residues tune the resonance between exciton 4–1 energy gap and an intramolecular chlorophyll vibration in reducing conditions to induce vibronic coupling and detune the resonance in oxidizing conditions. This redox-dependent modulation of the vibronic coupling steers excitations through different pathways in the complex to change the likelihood that they interact with exciton quenchers.     

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.     

IDO1 scavenges reactive oxygen species in myeloid-derived suppressor cells to prevent graft-versus-host disease

Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1^−/−) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1^−/− BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1^−/− Gr-1⁺CD11b⁺ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6C^lowLy6G^hi subset, compared with the WT counterparts. Importantly, Ido1^−/−Gr-1⁺CD11b⁺ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1^−/−Gr-1⁺CD11b⁺ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1^−/− BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1⁺CD11b⁺ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-binding metabolic enzyme that catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn). In addition to Trp catabolism, IDO1 has long been recognized to have immune-regulatory roles, preventing excessive inflammation (1). IDO1 is up-regulated in response to inflammatory stimuli, including Toll-like receptor (TLR) and type I/II interferon (IFN) signaling (1, 2). The induction of IDO1 after TLR9 stimulation has been demonstrated to mitigate experimental colitis (3). Catalytic function blockade in mice by pharmacological inhibition or genetic ablation of IDO1 (Ido1^−/−) enhanced inflammation and aggravated autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE) (4). The enhanced immune responses induced by IDO1 deficiency were associated with increased T helper (Th)1/Th17 responses; in contrast, regulatory T cell (Treg) responses were repressed (4–6). Consistently, IDO1 inhibition enhanced antitumor immune responses (7–9). The immune-regulatory effects of IDO1 have been ascribed to the depletion of Trp (10, 11) and the production of toxic catabolites along the Kyn pathway (4, 12–14). However, it remains unclear whether additional mechanisms are involved in IDO1-mediated immune suppression.Graft-versus-host disease (GVHD) is a severe inflammatory disease for which IDO1 has been shown to play a protective role (2, 14, 15). GVHD often develops as an adverse systemic complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is induced by activation of donor T cells reactive to the recipient’s major histocompatibility complexes (MHCs) and/or minor histocompatibility antigens (MiHAs) (16). Allo-reactivity of the activated donor T cells promotes tissue inflammation in the host, leading to morbidity and mortality. IDO1 deficiency in the bone marrow (BM) of the donor or the recipient has been linked to increased lethality (2, 14, 15), indicating a crucial role of IDO1 expression in the parenchymal and hematopoietic compartments in preventing GVHD. Kyn produced in IDO1-expressing lung epithelial cells and tissue macrophages suppressed T cell activation by binding to and activating immunomodulatory aryl hydrocarbon receptors (AhRs), which could explain the GVHD aggravation in Ido1^−/− recipients (14). Nevertheless, the mechanisms behind GVHD exacerbation by Ido1^−/− BM transfer remain obscure. Wild-type (WT) donor antigen-presenting cells prolonged survival in GVHD regardless of epithelial cell expression of IDO1, and IDO1 up-regulation after treatment of donor BM with TLR ligands reduced GVHD severity (2). These findings suggest an important role of IDO1 expressed by donor-derived myeloid cells in preventing severe GVHD. However, the immune-regulatory roles of IDO1 expressed in myeloid cells (termed myeloid IDO1 hereafter) remain elusive.Myeloid-derived suppressor cells (MDSCs) are innate cells that have immune-suppressive functions (17). Conventionally, MDSCs are identified as Gr-1⁺CD11b⁺ cells and can be further classified into Ly6C^hiLy6G^low monocytic (M) or Ly6C^lowLy6G^hi polymorphonuclear (PMN) subsets. MDSCs produce various immune-suppressive mediators, including arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and interleukin 10 (IL-10) (17, 18). Their ability to enhance Treg responses has also been reported (19, 20). As immature cells, MDSCs maintain the ability to differentiate into dendritic cells (DCs), macrophages, or neutrophils (21, 22). In GVHD, MDSCs derived from donor BM are the major population of myeloid cells expanding in the host (23), and along with Tregs they suppress GVHD (24–26). We previously reported that transplantation of MyD88-deficient (Myd88^−/−) BM suppressed Gr-1⁺CD11b⁺ cell expansion and polarized the differentiation of Gr-1⁺CD11b⁺ cells into DCs, aggravating GVHD (27, 28). These findings indicate that increasing the number of undifferentiated Gr-1⁺CD11b⁺ cells is essential for MDSC-mediated immune suppression in GVHD. Additionally, the finding that IDO1 expression in mononuclear cells, rather than in parenchymal cells, correlated positively with the survival of GVHD patients (29) suggested that IDOl expression in myeloid cells might be involved in the MDSC-mediated suppression of GVHD. Understanding the role of IDO1 in the function of MDSCs derived from the donor BM could lead to novel therapeutic strategies for the treatment of GVHD.In this study, we investigated the mechanisms underlying GVHD aggravation in hosts transplanted with IDO1-deficient BM. We found that IDO1 deficiency in donor BM did not affect the expansion of Gr-1⁺CD11b⁺ cells in GVHD hosts but polarized them toward a Ly6C^lowLy6G^hi phenotype, reducing their immune-regulatory potential. This phenomenon was ascribed to increased reactive oxygen species (ROS) generation in the Ido1^−/− Gr-1⁺CD11b⁺ cells and their skewing to neutrophil differentiation. Treatment of ROS-scavenging chemical reversed this phenomenon. Our findings suggest that the immune-regulatory roles of IDO1 are mediated by ROS scavenging and suppression of the differentiation of Gr-1⁺CD11b⁺ cells.