Search

Cherubism is a rare autosomal dominant disease caused mainly by a P416R point mutation of Src homology 3 Domain Binding Protein 2 (SH3BP2). Though macrophages and osteoblasts were thought to be the major contributors to the pathology in the past, the involvement of neutrophils in cherubism has been demonstrated by the increasing neutrophil infiltration and extensive neutrophil extracellular trap formation in the lesion recently. Here, we observed decreased expression of lineage marker Ly6G and impaired integrin activation on neutrophils, indicating a developmental defect of neutrophils in cherubism mice carrying the SH3BP2 P416R mutation. Furthermore, we performed single-cell RNA sequencing and compared transcriptomes between blood neutrophils, bone marrow neutrophils, and bone marrow leukocyte progenitors from wildtype and SH3BP2 P416R mice. RNA velocity and pathway analysis show that mutated neutrophils have stalled in the early stage of neutrophil development and failed to mature into granzyme pathway-enhanced, NET-formation-restrained mature neutrophil population in wildtype. Together, our study first illustrates the neutrophil development defects in cherubism as a mechanism for hyperinflammation characterized in cherubism. Ziming Cao and Keaton Karlinsey contributed equally. Beiyan Zhou, I-Ping Chen and Zhichao Fan are co-corresponding authors. This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454), and a startup fund from UConn Health. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Macrophages are central players in systemic inflammation associated with obesity and aging, termed meta-inflammation and inflammaging. Activities of macrophages elicited by the two chronic conditions display shared and distinct patterns mechanistically, resulting in multifaceted actions for their pathogenic roles. Drastically expanded tissue macrophage populations under obesity and aging stress attribute to both enhanced recruitment and local expansion. Importantly, molecular networks governing the multifaceted actions of macrophages are directly altered by environmental cues and subsequently contribute to metabolic reprogramming, resulting in meta-inflammation in obesity or inflammaging in aging. In this review, we will summarize how meta-inflammation and inflammaging affect macrophages and the molecular mechanisms involved in these processes.

Obesity is a prevalent health risk by inducing chronic, low-grade inflammation and insulin resistance, in part from adipose tissue inflammation perpetuated by activated B cells and other resident immune cells. However, regulatory mechanisms controlling B-cell actions in adipose tissue remain poorly understood, limiting therapeutic innovations. MicroRNAs are potent regulators of immune cell dynamics through fine-tuning a network of downstream genes in multiple signaling pathways. In particular, miR-150 is crucial to B-cell development and suppresses obesity-associated inflammation via regulating adipose tissue B-cell function. Herein, we review the effect of microRNAs on B-cell development, activation, and function and highlight miR-150-regulated B-cell actions during obesity which modulate systemic inflammation and insulin resistance. In this way, we hope to promote translational discoveries that mitigate obesity-induced health risks by targeting microRNA-regulated B-cell actions.

Macrophages are widely distributed immune cells that play central roles in a variety of physiologic and pathologic processes, including obesity and cardiovascular disease (CVD). They are highly plastic cells that execute diverse functions according to a combination of signaling and environmental cues. While macrophages have traditionally been understood to polarize to either proinflammatory M1-like or anti-inflammatory M2-like states, evidence has shown that they exist in a spectrum of states between those 2 phenotypic extremes. In obesity-related disease, M1-like macrophages exacerbate inflammation and promote insulin resistance, while M2-like macrophages reduce inflammation, promoting insulin sensitivity. However, polarization markers are expressed inconsistently in adipose tissue macrophages, and they additionally exhibit phenotypes differing from the M1/M2 paradigm. In atherosclerotic CVD, activated plaque macrophages can also exist in a range of proinflammatory or anti-inflammatory states. Some of these macrophages scavenge lipids, developing into heterogeneous foam cell populations. To better characterize the many actions of macrophages in human disease, we have designed a novel set of computational tools: MacSpectrum and AtheroSpectrum. These tools provide information on the inflammatory polarization status, differentiation, and foaming of macrophages in both human and mouse samples, allowing for better characterization of macrophage subpopulations based on their function. Using these tools, we identified disease-relevant cell states in obesity and CVD, including the novel concept that macrophage-derived foam cell formation can follow homeostatic noninflammatory or pathogenic inflammatory foaming programs.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT. This work was partially supported by the European Foundation for the Study of Diabetes (EFSD/Lilly, 2017 and EFSD/Boehringer Ingelheim European Research Programme on ‘‘Multi-System Challenges in Diabetes’’) and the Italian Ministry of Health (GR-2018-12367588) to D.L.-B.; Associazione Italiana per la Ricerca sul Cancro (AIRC) under IG 2019 - ID. 23562 project to K.A.; MIUR ‘‘Progetto Eccellenza’’ to Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita` degli Studi di Milano, and NUTRAGE (CNR FOE 2019, DSB.AD004.271) to A.S; Italian Foundation of Multiple Sclerosis (grant 2017/R/8), the Italian Ministry of Health (grant GR-2016-02362380) and the MAI Award grant to V. Chiurchiu; National Institutes of Health (NIH) common fund (DP5 OD028125) and the Burroughs Wellcome Fund (1019648) to J.R.B.; NIH K01DK125608 to F.S.; R01DK102898 and R01DK122808 to Y.- H.T.; and NIH RO1 DK121805 and AHA 19TPA34910079 to B.Z. A.H. was supported by RTI2018-095497-B-I00 from MICINN, HR17_00527 from La Caixa Foundation, and TNE-18CVD04 from the Leducq Foundation. M.S-D was supported by a fellowship PRE2019-08746 from the Ministerio de Ciencia e Innovacio´ n. M.R. was partially supported by a fellowship from AIRC (IG 2019 - ID. 23562) and by the Italian Ministry of Health (SG-2019-12368589). V.C. is part of the PhD Program in Evolutionary Biology and Ecology, Department of Biology, University of Rome Tor Vergata. Sí

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS “CD8+ TOXPHOS cells.” We validated that higher levels of OXPHOS in tumor- and peripheral blood–derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.

Background Immune checkpoint blockade (ICB) has greatly advanced the treatment of melanoma. A key component of ICB is the stimulation of CD8+ T cells in the tumor. However, ICB therapy only benefits a subset of patients and a reliable prediction method that does not require invasive biopsies is still a major challenge in the field. Methods We conducted a set of comprehensive single-cell transcriptomic analyses of CD8+ T cells in the peripheral blood (mPBL) and tumors (mTIL) from 8 patients with metastatic melanoma. Results Compared to circulating CD8+ T cells from healthy donors (hPBL), mPBLs contained subsets resembling certain features of mTIL. More importantly, three clusters (2, 6 and 15) were represented in both mPBL and mTIL. Cluster 2 was the major subset of the majority of hPBL, which phenocopied hallmark parameters of resting T cells. Cluster 6 and 15 were uniquely presented in melanoma patients. Cluster 15 had the highest PD-1 levels, with elevated markers of both activation and dysfunction/exhaustion; while Cluster 6 was enriched for ‘dormant’ cells with overall toned-down transcriptional activity except PPAR signaling, a known suppressor for T cell activation. Interestingly, unlike other mTIL clusters that would classically be defined as exhausted, Cluster 15 exhibited the highest metabolic activity (oxidative-phosphorylation and glycolysis). We further analyzed total sc-transcriptomics using cell trajectory algorithms and identified that these three clusters were the most distinct subtypes of CD8 T cells from each other, representing: resting (cluster 2), metabolically active-dysfunctional (cluster 15), and dormant phenotypes (cluster 6). Further, three unique intracellular programs in melanoma drive the transition of resting CD8+ T cells (cluster 2) to both metabolic/dysfunctional (cluster 15) and dormant states (cluster 6) that are unique to tumor bearing conditions. Based on these high-resolution analyses, we developed original algorithms to build a novel ICB response predictive model using immune-blockade co-expression gene patterns. The model was trained and tested using previously published GEO datasets containing CD8 T cells from anti-PD-1 treated patients and presented an AUC of 0.82, with 92% and 89% accuracy of ICB response in the two datasets. Conclusions We identified and analyzed unique populations of CD8+ T cells in circulation and tumor using high-resolution single-cell transcriptomics to define the landscape of CD8+ T cell states, revealing critical subsets with shared features in PBLs and TILs. Most importantly, we established an innovative model for ICB response prediction by using peripheral blood lymphocytes. Ethics Approval This study was performed under an IRB approved protocol.

Schwann cells play a major role in helping heal injured nerves. They help clear debris, produce neurotrophins, upregulate neurotrophin receptors, and form bands of Bungner to guide the healing nerve. But nerves do not always produce enough neurotrophins and neurotrophin receptors to repair themselves. Nerve growth factor (NGF) is an important neurotrophin for promoting nerve healing and lysophosphatidylcholine (LPC) has been shown to stimulate NGF receptors (NGFR). This study tested the administration of a single intraneural injection of LPC (1 mg/mL for single LPC injection and 10 mg/mL for multiple LPC injections) at day 0 and one (day 7), two (days 5 and 7), or three (days 5, 7, and 9) injections of NGF (160 ng/mL for single injections and 80 ng/mL for multiple injections) to determine baseline effects on crushed sciatic nerves in rats. The rats were randomly divided into four groups: control, crush, crush-NGF, and crush-LPC-NGF. The healing of the nerves was measured weekly by monitoring gait; electrophysiological parameters: compound muscle action potential (CMAP) amplitudes; and morphological parameters: total fascicle areas, myelinated fiber counts, fiber densities, fiber packing, and mean g-ratio values at weeks 3 and 6. The crush, crush-NGF, and crush-LPC-NGF groups statistically differed from the control group for all six weeks for the electrophysiological parameters but only differed from the control group at week 3 for the morphological parameters. The crush, crush-NGF, and crush-LPC-NGF groups did not differ from each other over the course of the study. Single injections of LPC and NGF one week apart or multiple treatments of NGF at 5, 7 and 9 days post-injury did not alter the healing rate of the sciatic nerves during weeks 1-6 of the study. These findings are important to define the baseline effects of NGF and LPC injections, as part of a larger effort to determine the minimal dose regimen of NGF to regenerate peripheral nerves.

Long non-coding RNAs (lncRNAs) are critical regulators of immune responses, but their role in sepsis is largely unknown. During sepsis, TLR sensing of microbes triggers the “cytokines storm”, disseminated intravascular coagulation and organ damage. Patients surviving systemic inflammatory response syndrome develop profound immunosuppression that resembles TLR tolerance and often succumb to secondary infections. To study the sepsis-associated “cytokine storm”, we used in vivo exposure of mice to LPS (endotoxemia) and Pam3Cys, while endotoxin tolerization of macrophages in vitro was employed to examine features of sepsis-associated immunosuppression. This study showed that LPS-treated macrophages decreased expression of lincRNA-EPS while upregulating lincRNA-Cox2 levels. Using knockout mice, we demonstrated that lincRNA-EPS acts to restrain KC, IL6 and CCL5 expression in the serum, spleen, lungs and liver of LPS or Pam3Cys-treated mice. Infection of lincRNA-EPS−/− macrophages with Klebsiella pneumoniae led to increased expression of IL-6 and CCL5 compared to wild-type cells. K. pneumoniae-infected wild-type and lincRNA-EPS−/− macrophages exhibited similar levels of TNF-α and bacterial 16S RNA. Compared to wild-type animals, pulmonary K. pneumoniae infection of lincRNA-EPS−/− mice resulted in higher levels of serum and lung IL-6 but decreased lung TNF-α. Finally, we showed that lincRNA-EPS and lincRNA-Cox2 are not required for LPS tolerance induction in macrophages. Thus, lincRNA-EPS and lincRNA-Cox2 shape macrophage-elicited cytokine induction upon exposure to microbial agonists and regulate host responses to K. pneumoniae.