Adrenal gland macrophages regulate glucocorticoid production through Trem2 and TGFß.

Glucocorticoid synthesis by adrenal glands (AG) is regulated by the hypothalamic-pituitary-adrenal axis (HPA-axis) to facilitate stress responses when the host is exposed to stimuli. Recent studies have implicated macrophages (MФ) as potential steroidogenic regulators, but the molecular mechanisms by which AG MФ exert such influence remain unclear. In this study, we investigated the role of AG MФ in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Utilizing single-cell RNA sequencing, we observed dynamic AG MФ polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among the transcriptional alterations induced in MФ, Triggering Receptor Expressed on Myeloid (Trem2) was highlighted due to its dramatic upregulation following stress. Conditional deletion of MФ Trem2 revealed a protective role for Trem2 in stress responses. Mechanistically, Trem2 deletion led to increased AG MФ death, abolished the TGFß-producing capacity of AG MФ, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGFß signaling. Together, these observations suggest that AG MФ restrict steroidogenesis through Trem2 and TGFß, which opens potential avenues for immunotherapeutic interventions targeting the innate immune system to resolve stress-related disorders.

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability-Brief Report.

BACKGROUND: Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. METHODS: To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. RESULTS: AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. CONCLUSIONS: Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.

Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) orchestrates a suppressive tumor microenvironment that fosters immunotherapy resistance. Tumor-associated macrophages (TAMs) are the principal immune cell infiltrating PDA and are heterogeneous. Here, by employing macrophage fate-mapping approaches and single-cell RNA sequencing, we show that monocytes give rise to most macrophage subsets in PDA. Tumor-specific CD4, but not CD8, T cells promote monocyte differentiation into MHCIIhi anti-tumor macrophages. By conditional major histocompatibility complex (MHC) class II deletion on monocyte-derived macrophages, we show that tumor antigen presentation is required for instructing monocyte differentiation into anti-tumor macrophages, promoting Th1 cells, abrogating Treg cells, and mitigating CD8 T cell exhaustion. Non-redundant IFNγ and CD40 promote MHCIIhi anti-tumor macrophages. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from that of tissue-resident macrophages following loss of macrophage MHC class II or tumor-specific CD4 T cells. Thus, tumor antigen presentation by macrophages to CD4 T cells dictates TAM fate and is a major determinant of macrophage heterogeneity in cancer.

FFAR4 regulates cardiac oxylipin balance to promote inflammation resolution in HFpEF secondary to metabolic syndrome.

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome, but a predominant subset of HFpEF patients has metabolic syndrome (MetS). Mechanistically, systemic, nonresolving inflammation associated with MetS might drive HFpEF remodeling. Free fatty acid receptor 4 (Ffar4) is a GPCR for long-chain fatty acids that attenuates metabolic dysfunction and resolves inflammation. Therefore, we hypothesized that Ffar4 would attenuate remodeling in HFpEF secondary to MetS (HFpEF-MetS). To test this hypothesis, mice with systemic deletion of Ffar4 (Ffar4KO) were fed a high-fat/high-sucrose diet with L-NAME in their water to induce HFpEF-MetS. In male Ffar4KO mice, this HFpEF-MetS diet induced similar metabolic deficits but worsened diastolic function and microvascular rarefaction relative to WT mice. Conversely, in female Ffar4KO mice, the diet produced greater obesity but no worsened ventricular remodeling relative to WT mice. In Ffar4KO males, MetS altered the balance of inflammatory oxylipins systemically in HDL and in the heart, decreasing the eicosapentaenoic acid-derived, proresolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE), while increasing the arachidonic acid-derived, proinflammatory oxylipin 12-hydroxyeicosatetraenoic acid (12-HETE). This increased 12-HETE/18-HEPE ratio reflected a more proinflammatory state both systemically and in the heart in male Ffar4KO mice and was associated with increased macrophage numbers in the heart, which in turn correlated with worsened ventricular remodeling. In summary, our data suggest that Ffar4 controls the proinflammatory/proresolving oxylipin balance systemically and in the heart to resolve inflammation and attenuate HFpEF remodeling.

Posttransplantation cyclophosphamide expands functional myeloid-derived suppressor cells and indirectly influences Tregs.

Posttransplantation cyclophosphamide (PTCy), given on days +3 and +4, reduces graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but its immunologic underpinnings are not fully understood. In a T-cell-replete, major histocompatibility complex-haploidentical murine HCT model (B6C3F1→B6D2F1), we previously showed that PTCy rapidly induces suppressive mechanisms sufficient to prevent GVHD induction by non-PTCy-exposed donor splenocytes infused on day +5. Here, in PTCy-treated mice, we found that depleting Foxp3+ regulatory T cells (Tregs) in the initial graft but not the day +5 splenocytes did not worsen GVHD, yet depleting Tregs in both cellular compartments led to fatal GVHD induced by the day +5 splenocytes. Hence, Tregs were necessary to control GVHD induced by new donor cells, but PTCy's impact on Tregs appeared to be indirect. Therefore, we hypothesized that myeloid-derived suppressor cells (MDSCs) play a complementary role. Functionally suppressive granulocytic and monocytic MDSCs were increased in percentages in PTCy-treated mice, and MDSC percentages were increased after administering PTCy to patients undergoing HLA-haploidentical HCT. PTCy increased colony-stimulating factors critical for MDSC development and rapidly promoted the generation of MDSCs from bone marrow precursors. MDSC reduction via anti-Gr1 treatment in murine HCT did not worsen histopathologic GVHD but resulted in decreased Tregs and inferior survival. The clinical implications of these findings, including the potential impact of expanded MDSCs after PTCy on engraftment and cytokine release syndrome, remain to be elucidated. Moreover, the indirect effect that PTCy has on Tregs, which in turn play a necessary role in GVHD prevention by initially transplanted or subsequently infused T cells, requires further investigation.

Murine allogeneic CAR T cells integrated before or early after posttransplant cyclophosphamide exert antitumor effects.

Relapse limits the therapeutic efficacy both of chimeric antigen receptor (CAR) T cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the 2 therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR T cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after posttransplant cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR T cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert antitumor effects. Indeed, allogeneic anti-CD19 CAR T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR T-cell treatment on day +9 or day +14 was safe but less effective, suggesting a limited therapeutic window. CAR T cells infused before PTCy were not eliminated, but surviving CAR T cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR T-cell expansion, higher phenotypic CAR T-cell activation, less CD4+CD25+Foxp3+ CAR T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR T cells and more cytotoxic CD8+ CAR T cells. This study provides mechanistic insight into PTCy's impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR T cells and allo-HCT that may compensate for deficiencies of each individual approach.

Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis.

Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.

Efflux capacity and aldehyde dehydrogenase both contribute to CD8+ T-cell resistance to posttransplant cyclophosphamide.

Mechanisms of T-cell survival after cytotoxic chemotherapy, including posttransplantation cyclophosphamide (PTCy), are not well understood. Here, we explored the impact of PTCy on human CD8+ T-cell survival and reconstitution, including what cellular pathways drive PTCy resistance. In major histocompatibility complex (MHC)-mismatched mixed lymphocyte culture (MLC), treatment with mafosfamide, an in vitro active cyclophosphamide analog, preserved a relatively normal distribution of naïve and memory CD8+ T cells, whereas the percentages of mucosal-associated invariant T (MAIT) cells and phenotypically stem cell memory (Tscm) T-cell subsets were increased. Activated (CD25+) and proliferating CD8+ T cells were derived from both naïve and memory subsets and were reduced but still present after mafosfamide. By contrast, cyclosporine-A (CsA) or rapamycin treatment preferentially maintained nonproliferating CD25- naïve cells. Drug efflux capacity and aldehyde dehydrogenase-1A1 expression were increased in CD8+ T cells in allogeneic reactions in vitro and in patients, were modulated by common γ-chain cytokines and the proliferative state of the cell, and contributed to CD8+ T-cell survival after mafosfamide. The CD8+ T-cell composition early after hematopoietic cell transplantation (HCT) in PTCy-treated patients was dominated by CD25+ and phenotypically memory, including Tscm and MAIT, cells, consistent with MLC. Yet, MHC-mismatched murine HCT studies revealed that peripherally expanded, phenotypically memory T cells 1 to 3 months after transplant originated largely from naïve-derived rather than memory-derived T cells surviving PTCy, suggesting that initial resistance and subsequent immune reconstitution are distinct. These studies provide insight into the complex immune mechanisms active in CD8+ T-cell survival, differentiation, and reconstitution after cyclophosphamide, with relevance for post-HCT immune recovery, chemotherapy use in autologous settings, and adoptive cellular therapies.

Distinct myeloid antigen-presenting cells dictate differential fates of tumor-specific CD8+ T cells in pancreatic cancer.

We investigate how myeloid subsets differentially shape immunity to pancreatic ductal adenocarcinoma (PDA). We show that tumor antigenicity sculpts myeloid cell composition and functionality. Antigenicity promotes accumulation of type 1 dendritic cells (cDC1), which is driven by Xcr1 signaling, and overcomes macrophage-mediated suppression. The therapeutic activity of adoptive T cell therapy or programmed cell death ligand 1 blockade required cDC1s, which sustained splenic Klrg1+ cytotoxic antitumor T cells and functional intratumoral T cells. KLRG1 and cDC1 genes correlated in human tumors, and PDA patients with high intratumoral KLRG1 survived longer than patients with low intratumoral KLRG1. The immunotherapy CD40 agonist also required host cDC1s for maximal therapeutic benefit. However, CD40 agonist exhibited partial therapeutic benefit in cDC1-deficient hosts and resulted in priming of tumor-specific yet atypical CD8+ T cells with a regulatory phenotype and that failed to participate in tumor control. Monocyte/macrophage depletion using clodronate liposomes abrogated T cell priming yet enhanced the antitumor activity of CD40 agonist in cDC1-deficient hosts via engagement of innate immunity. In sum, our study supports that cDC1s are essential for sustaining effective antitumor T cells and supports differential roles for cDC1s and monocytes/macrophages in instructing T cell fate and immunotherapy response.

Metabolic regulation of macrophage proliferation and function in atherosclerosis.

PURPOSE OF REVIEW: Macrophage accumulation within atherosclerotic plaque is a primary driver of disease progression. However, recent advances in both phenotypic and functional heterogeneity of these cells have allowed for improved insight into potential regulation of macrophage function within lesions. In this review, we will discuss recent insights on macrophage heterogeneity, lipid processing, metabolism, and proliferation in atherosclerosis. Furthermore, we will identify outstanding questions in the field that are pertinent to future studies. RECENT FINDINGS: With the recent development of single-cell RNA sequencing, several studies have highlighted the diverse macrophage populations within plaques, including pro-inflammatory, anti-inflammatory, lipid loaded and tissue resident macrophages. Furthermore, new data has suggested that differential activation of metabolic pathways, including glycolysis and fatty acid oxidation, may play a key role in determining function. Recent works have highlighted that different populations retain varying capacity to undergo proliferation; regulating the proliferation pathway may be highly effective in reducing plaque in advanced lesions. SUMMARY: Macrophage populations within atherosclerosis are highly heterogeneous; differences in cytokine production, lipid handling, metabolism, and proliferation are seen between subpopulations. Understanding the basic cellular mechanisms that drive this heterogeneity will allow for the development of highly specific disease modulating agents to combat atherosclerosis.

Optimized Timing of Post-Transplantation Cyclophosphamide in MHC-Haploidentical Murine Hematopoietic Cell Transplantation.

Post-transplantation cyclophosphamide (PTCy) reduces the risks of severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Yet, the standard clinical dose and timing of PTCy were partly extrapolated from MHC-matched skin allografting models and were partly empirical. Here we investigated the impact of differential dosing and timing of PTCy on its efficacy in preventing GVHD in a murine MHC-haploidentical HCT model. Administration of PTCy on days +3/+4 was superior to administration on days +1/+2, +5/+6, or +7/+8, whereas low-dose (10 mg/kg/day) PTCy on days +1/+2 actually led to accelerated death. Although the optimal timing of PTCy dosing was day +2 or +3 in the skin allografting models, in our MHC-haploidentical HCT model, PTCy on days +2/+3 was inferior to PTCy on days +3/+4 at lower doses. PTCy administered on days +3/+4, +4/+5, or +3/+5 were similarly efficacious. Single-day versus 2-day dosing schedules demonstrated that PTCy is maximally effective when given on day +4. Flow cytometric analysis showed that optimal PTCy dosing schedules both decreased alloreactive CD4+CD25-Foxp3- T cell proliferation at day +7 and allowed preferential CD4+CD25+Foxp3+ T cell reconstitution at day +21, suggesting that this combination may be a potential predictive biomarker of successful GVHD prevention by PTCy. These results show that the dose, timing, and cumulative exposure of PTCy all are critical for its efficacy in preventing GVHD. We are currently investigating the clinical relevance of these findings in a protocol seeking to optimize PTCy dose and timing and test these T cell endpoints as candidate biomarkers of successful GVHD prevention by PTCy.

Post-transplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression.

Post-transplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet, our understanding of how PTCy prevents graft-versus-host disease (GVHD) largely has been extrapolated from major histocompatibility complex (MHC)-matched murine skin allografting models that were highly contextual in their efficacy. Herein, we developed a T-cell-replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) to test the putative underlying mechanisms: alloreactive T-cell elimination, alloreactive T-cell intrathymic clonal deletion, and suppressor T-cell induction. In this model and confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific Vßs or the 2C or 4C T-cell receptors. Furthermore, the thymus was not necessary for PTCy's efficacy. Rather, PTCy induced alloreactive T-cell functional impairment which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of CD4+CD25+Foxp3+ regulatory T cells, including those that were alloantigen-specific, which served an increasingly critical function over time. Our results prompt a paradigm-shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes.

Long-term Effectiveness of Trabectome (Ab-interno Trabeculectomy) Surgery.

AIM: To evaluate the long-term safety and efficacy of ab-interno trabeculectomy with trabectome for the treatment of glaucoma. MATERIALS AND METHODS: Data collected for 339 eyes which included demographics, intraocular pressure (IOP) measurements using Goldmann applanation tonometry, best-corrected visual acuity (BCVA), visual field results, optic nerve status, gonioscopic findings, prior glaucoma procedures, number of glaucoma medications and pain level. The main data points of interest were preoperative IOP vs. postoperative IOP and BCVA, medication use, pain status, and complications. RESULTS: Of the 339 eyes that underwent trabectome, we found a statistically significant reduction in IOP (p < 0.01) at final follow-up (average = 18.35 months) of nearly 23%, with a complication rate of 5.86%. Furthermore, this reduction was maintained up to 8 years post procedure. LogMAR visual acuity was significantly improved in 69% of eyes at the final visit (p < 0.05), while only 1.77% of cases saw a significant reduction. Based on these findings, we determined a success rate of around 80% to 100 months following trabectome. CONCLUSION: Trabectome is a safe and effective long term for most forms and severities of glaucoma. HOW TO CITE THIS ARTICLE: Bendel RE, Patterson MT. Long-term Effectiveness of Trabectome (Ab-interno Trabeculectomy) Surgery. J Curr Glaucoma Pract 2018;12(3):119-124.