Protein kinase C delta regulates mononuclear phagocytes and hinders response to immunotherapy in cancer.

Mononuclear phagocytes (MPs) play a crucial role in tissue homeostasis; however, MPs also contribute to tumor progression and resistance to immune checkpoint blockade (ICB). Targeting MPs could be an effective strategy to enhance ICB efficacy. We report that protein kinase C delta (PKCδ), a serine/threonine kinase, is abundantly expressed by MPs in human and mouse tumors. PKCδ-/- mice displayed reduced tumor progression compared to wild types, with increased response to anti-PD-1. Tumors from PKCδ-/- mice demonstrated TH1-skewed immune response including increased antigen presentation and T cell activation. Depletion of MPs in vivo altered tumor growth in control but not PKCδ-/- mice. Coinjection of PKCδ-/- M2-like macrophages with cancer cells into wild-type mice markedly delayed tumor growth and significantly increased intratumoral T cell activation compared to PKCδ+/+ controls. PKCδ deficiency reprogrammed MPs by activating type I and type II interferon signaling. Thus, PKCδ might be targeted to reprogram MPs to augment ICB efficacy.

Dynamics of horizontal walking and vertical climbing in the Australian green tree frog (Ranoidea caerulea).

Despite the high mechanical demands associated with climbing, the ability to ascend vertically has evolved independently in most major animal lineages. However, little is known about the kinetics, mechanical energy profiles or spatiotemporal gait characteristics of this locomotor mode. In this study, we explored the dynamics of horizontal locomotion and vertical climbing on both flat substrates and narrow poles in five Australian green tree frogs (Ranoidea caerulea). Vertical climbing is associated with slow, deliberate movements (i.e. reduced speed and stride frequency and increased duty factors) and propulsive fore-aft impulses in both the forelimb and hindlimb. By comparison, horizontal walking was characterized by a braking forelimb and a propulsive hindlimb. In the normal plane, tree frogs mirrored other taxa in exhibiting a net pulling forelimb and a net pushing hindlimb during vertical climbing. In terms of mechanical energy, tree frogs matched theoretical predictions of climbing dynamics (i.e. the total mechanical energetic cost of vertical climbing was predominantly driven by potential energy, with negligible kinetic contributions). Utilizing power as a means of estimating efficiency, we also demonstrate that Australian green tree frogs show total mechanical power costs only slightly above the minimum mechanical power necessary to climb, highlighting their highly effective locomotor mechanics. This study provides new data on climbing dynamics in a slow-moving arboreal tetrapod and raises new testable hypotheses about how natural selection can act upon a locomotor behavior that is notably constrained by external physical forces.

Three toes and three modes: Dynamics of terrestrial, suspensory, and vertical locomotion in brown-throated three-toed sloths (Bradypodidae, Xenarthra).

Living sloths exhibit numerous anatomical specializations towards inverted quadrupedalism, however, previous studies have noted a more varied locomotor repertoire than previously anticipated. In this study, we present spatiotemporal gait characteristics and triaxial kinetic data from the brown-throated three-toed sloth (Bradypus variegatus) across three locomotor modes: terrestrial quadrupedal "crawling", suspensory walking, and vertical climbing. Compared to quadrupedal crawling and suspensory walking, B. variegatus adopted longer contact times and stride durations, larger duty factors, and greater speed during vertical climbing. Net fore-aft impulses were significantly greater during vertical climbing in both limb pairs than in quadrupedal crawling and suspensory walking. Functionally, during quadrupedal crawling and vertical climbing, both limb pairs served propulsive roles, while differentiation between a propulsive forelimb and braking hindlimb was observed during suspension. Net tangential forces differentiated vertical climbing kinetics from the other modes of locomotion, with the introduction of bidirectional pulling and pushing forces in the forelimb and hindlimb, respectively. The net mediolateral impulses were similar in vertical climbing and quadrupedal crawling as both limb pairs directed forces in one direction, whereas during suspensory walking, the laterally dominant forelimb was opposed by the medially dominant hindlimb. In total, this study provides novel data on the diverse locomotor dynamics in a slow-moving arboreal tetrapod and posits new testable hypotheses about the neuroplasticity and ease of transitioning between locomotor behaviors. The strikingly similar kinetic profiles of quadrupedal crawling and suspensory walking compared to vertical climbing suggest shared neuromuscular and mechanical demands between these mirrored locomotor modes.

PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8+ T cells in breast cancer.

Myeloid-derived suppressor cells (MDSCs) are an immature innate cell population that expands in pathological conditions such as cancer and suppresses T cells via production of immunosuppressive factors. Conversely, efficient cytotoxic T cell priming is dependent on the ability of antigen-presenting cells (APCs) to cross-present tumor antigens to CD8+ T cells, a process that requires a specific subtype of dendritic cells (DCs) called conventional DC1 (cDC1) which are often dysfunctional in cancer. One way to activate cDC1 is ligation of CD40 which is abundantly expressed by myeloid cells and its agonism leads to myeloid cell activation. Thus, targeting MDSCs while simultaneously expanding cross-presenting DCs represents a promising strategy that, when combined with agonistic CD40, may result in long-lasting protective immunity. In this study, we investigated the effect of PKC agonists PEP005 and prostratin on MDSC expansion, differentiation, and recruitment to the tumor microenvironment. Our findings demonstrate that PKC agonists decreased MDSC expansion from hematopoietic progenitors and induced M-MDSC differentiation to an APC-like phenotype that expresses cDC1-related markers via activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Simultaneously, PKC agonists favored cDC1 expansion at the expense of cDC2 and plasmacytoid DCs (pDC). Functionally, PKC agonists blunted MDSC suppressive activity and enhanced MDSC cross-priming capacity both in vitro and in vivo. Finally, combination of PKC agonism with agonistic CD40 mAb resulted in a marked reduction in tumor growth with a significant increase in intratumoral activated CD8+ T cells and tissue-resident memory CD8+ T cells in a syngeneic breast cancer mouse model. In sum, this work proposes a novel promising strategy to simultaneously target MDSCs and promote APC function that may have highly impactful clinical relevance in cancer patients.

Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 Spike Protein Subunit 1.

A hallmark of COVID-19 is a hyperinflammatory state associated with severity. Monocytes undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism associated with production of pro-inflammatory cytokines. This response was dependent on hypoxia-inducible factor-1α, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production and metabolic reprogramming. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.

Immune checkpoint blockade reprograms systemic immune landscape and tumor microenvironment in obesity-associated breast cancer.

Immune checkpoint blockade (ICB) has improved outcomes in some cancers. A major limitation of ICB is that most patients fail to respond, which is partly attributable to immunosuppression. Obesity appears to improve immune checkpoint therapies in some cancers, but impacts on breast cancer (BC) remain unknown. In lean and obese mice, tumor progression and immune reprogramming were quantified in BC tumors treated with anti-programmed death-1 (PD-1) or control. Obesity augments tumor incidence and progression. Anti-PD-1 induces regression in lean mice and potently abrogates progression in obese mice. BC primes systemic immunity to be highly responsive to obesity, leading to greater immunosuppression, which may explain greater anti-PD-1 efficacy. Anti-PD-1 significantly reinvigorates antitumor immunity despite persistent obesity. Laminin subunit beta-2 (Lamb2), downregulated by anti-PD-1, significantly predicts patient survival. Lastly, a microbial signature associated with anti-PD-1 efficacy is identified. Thus, anti-PD-1 is highly efficacious in obese mice by reinvigorating durable antitumor immunity. VIDEO ABSTRACT.

Growth differentiation factor-15 is associated with age-related monocyte dysfunction.

OBJECTIVE: Age-associated decreases in immune functions are precipitated by a variety of mechanisms and affect nearly every immune cell subset. In myeloid cells, aging reduces numbers of phagocytes and impairs their functional abilities, including antigen presentation, phagocytosis, and bacterial clearance. Recently, we described an aging effect on several functions in monocytes, including impaired mitochondrial function and reduced inflammatory cytokine gene expression during stimulation with lipopolysaccharide. We hypothesized that circulating factors altered by the aging process underly these changes. Growth differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor-ß superfamily that has known anti-inflammatory effects in macrophages and has been shown to be highly differentially expressed during aging. METHODS: We used biobanked plasma samples to assay circulating GDF-15 levels in subjects from our previous studies and examined correlations between GDF-15 and monocyte function. RESULTS: Monocyte interleukin-6 production due to lipopolysaccharide stimulation was negatively correlated to plasma GDF-15. Additionally, GDF-15 was positively correlated to circulating CD16 + monocyte proportions and negatively correlated to monocyte mitochondrial respiratory capacity. CONCLUSIONS: These results suggest that GDF-15 is a potential circulating factor affecting a variety of monocyte functions and promoting monocyte immunosenescence and thus may be an attractive candidate for therapeutic intervention to ameliorate this.

Macrophage Immunometabolism and Inflammaging: Roles of Mitochondrial Dysfunction, Cellular Senescence, CD38, and NAD.

Aging is a complex process that involves dysfunction on multiple levels, all of which seem to converge on inflammation. Macrophages are intimately involved in initiating and resolving inflammation, and their dysregulation with age is a primary contributor to inflammaging-a state of chronic, low-grade inflammation that develops during aging. Among the age-related changes that occur to macrophages are a heightened state of basal inflammation and diminished or hyperactive inflammatory responses, which seem to be driven by metabolic-dependent epigenetic changes. In this review article we provide a brief overview of mitochondrial functions and age-related changes that occur to macrophages, with an emphasis on how the inflammaging environment, senescence, and NAD decline can affect their metabolism, promote dysregulation, and contribute to inflammaging and age-related pathologies.

Classical monocytes from older adults maintain capacity for metabolic compensation during glucose deprivation and lipopolysaccharide stimulation.

Inflammaging is the chronic low-grade inflammation that occurs with age that contributes to the pathology of age-related diseases. Monocytes are innate immune cells that become dysregulated with age and which can contribute to inflammaging. Metabolism plays a key role in determining immune cell functions, with anti-inflammatory cells primarily relying on fatty acid oxidation and pro-inflammatory cells primarily relying on glycolysis. It was recently shown that lipopolysaccharide (LPS)-stimulated monocytes can compensate for a lack of glucose by utilizing fatty acid oxidation. Given that mitochondrial function decreases with age, we hypothesized that classical monocytes taken from aged individuals would have an impaired ability to upregulate oxidative metabolism along with impaired effector functions. Aging did not impair LPS-induced oxygen consumption rate during glucose deprivation as measured on a Seahorse XFp system. Additionally, aged classical monocytes maintained inflammatory gene expression responses and phagocytic capacity during LPS stimulation in the absence of glucose. In conclusion, aged classical monocytes maintain effector and metabolic functions during glucose deprivation, at least in an ex vivo context.

Classical monocytes maintain ex vivo glycolytic metabolism and early but not later inflammatory responses in older adults.

BACKGROUND: Inflammaging is a condition of chronic low-grade inflammation due to the aging process and is associated with a variety of chronic diseases. Monocytes are innate immune cells which contribute to inflammation and are dysregulated during aging, demonstrated reduced phagocytosis, increased inflammation, and alterations in subset proportions. Metabolism is known to determine immune cell function, with quiescent and anti-inflammatory cells primarily relying on fatty acid oxidation, while activated and inflammatory cells primarily rely on glycolysis. We have previously shown an age-related decrease in mitochondrial respiratory capacity in monocytes, so we hypothesized here that a compensatory shift toward glycolysis would occur which would also exacerbate inflammation. RESULTS: Using Seahorse assays, we profiled glycolysis in classical monocytes isolated from older (60-80 yr) and younger (18-35 yr) adults. Aging did not affect parameters of basal glycolysis in the glycolysis stress test, nor did it alter glycolytic activation early (2 h) or later (24 h) post-LPS stimulation. Cytokine gene expression was unchanged between aged and young subjects at 2 h post-LPS but was reduced in older subjects at 24 h post-LPS either significantly (IL1B) or near-significantly (IL6, IL10). CONCLUSIONS: Aging appears not to affect glycolytic metabolism ex vivo in classical monocytes, but may reduce cytokine expression at later timepoints. Studies examining monocytes stimulated with age-altered circulating factors or with other pattern recognition receptor agonists may shed further light on monocyte metabolism as a determinant of immunosenescence and inflammaging.

Aging impairs mitochondrial respiratory capacity in classical monocytes.

Aging is a critical healthcare concern, with age-related inflammation disposing individuals to a variety of diseases. Monocytes are affected by the aging process, with increased inflammation and impaired cellular functions such as phagocytosis. Mechanisms by which aging alters monocyte function are unknown, but recent research suggests that the balance of metabolic processes determine immune cell phenotype and function. Given the known association between aging and mitochondrial dysfunction in other tissues, we hypothesized that aging would impair mitochondrial function in monocytes. To test this, we isolated classical monocytes from young and older adults and tested mitochondrial function by a Seahorse assay. Aging reduced mitochondrial respiratory capacity and spare capacity in monocytes. Mitochondrial dysfunction is a potential mechanism by which aging alters monocyte phenotype and may impair inflammatory functions, especially in low-glucose environments where oxidative metabolism is necessary to meet energy demands.