Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

Dose-dependent effects of red blood cell transfusion and case mix index on venous thromboembolic events in spine surgery.

BACKGROUND AND OBJECTIVES: Venous thromboembolic (VTE) events represent a major source of morbidity and mortality in spine surgery. Our goal was to assess whether a dose-response relationship exists between red blood cell (RBC) transfusion and postoperative VTE events among spine surgery patients. MATERIALS AND METHODS: A total of 786 spine surgery patients at a single institution who received at least 1 RBC unit perioperatively were included (2016-2019). Patients were stratified based on RBC transfusion volume: 1-2 units (39.3%), 3-4 units (29.4%), 5-6 units (15.9%) and ≥7 units (15.4%). Subgroup analyses were performed after stratification by case mix index, a standardized surrogate for patients' disease severity and comorbidities. Multivariable regression was used to assess risk factors for the development of postoperative VTE events. RESULTS: The overall VTE event rate was 2.4% (n = 19). A dose-response relationship was seen between RBC transfusion volume and VTE events (1-2 units: 0.97%, 3-4 units: 1.30%, 5-6 units: 3.20%, ≥7 units: 7.44%; p < 0.01). Similar dose-response relationships were seen between case mix index and VTE events (1.00-3.99: 0.52%, 4.00-6.99: 2.68%, ≥7.00: 9.00%; p < 0.01). On multivariable regression, larger RBC transfusion volumes (adjusted odds ratio [OR] 1.18 per RBC unit, 95% confidence interval [CI] 1.07-1.29; p < 0.01) and higher case mix index scores (adjusted OR 1.39 per unit increase, 95% CI 1.14-1.69; p < 0.01) were associated with an increased risk of thrombosis. CONCLUSION: Larger RBC transfusion volumes and higher case mix index scores were associated with an increased risk of VTE events. Physicians should be aware of how these dose-response relationships can influence a patient's risk of developing thrombotic complications postoperatively.

Efferocytosis by Paneth cells within the intestine.

Apoptotic cells are quickly and efficiently engulfed and removed via the process of efferocytosis by either professional phagocytes, such as macrophages, or non-professional phagocytes, including epithelial cells.1,2 In addition to debris removal, a key benefit of efferocytosis is that phagocytes engulfing apoptotic cells release anti-inflammatory mediators3,4 that help reduce local tissue inflammation;5 conversely, accumulation of uncleared apoptotic cells predisposes to a pro-inflammatory tissue milieu.6-8 Due to their high proliferative capacity, intestinal epithelial cells (iECs) are sensitive to inflammation, irradiation, and chemotherapy-induced DNA damage, leading to apoptosis. Mechanisms of iEC death in the context of irradiation has been studied,9,10 but phagocytosis of dying iECs is poorly understood. Here, we identify an unexpected efferocytic role for Paneth cells, which reside in intestinal crypts and are linked to innate immunity and maintenance of the stem cell niche in the crypt.11,12 Through a series of studies spanning in vitro efferocytosis, ex vivo intestinal organoids ("enteroids"), and in vivo Cre-mediated deletion of Paneth cells, we show that Paneth cells mediate apoptotic cell uptake of dying neighbors. The relevance of Paneth-cell-mediated efferocytosis was revealed ex vivo and in mice after low-dose cesium-137 (137Cs) irradiation, mimicking radiation therapies given to cancer patients often causing significant apoptosis of iECs. These data advance a new concept that Paneth cells can act as phagocytes and identify another way in which Paneth cells contribute to the overall health of the intestine. These observations also have implications for individuals undergoing chemotherapy or chronic inflammatory bowel disease.

Abdominal Pain and Intermittent Fevers in a 16-Year-Old Girl.

A 16-year-old girl presented to the emergency department with intermittent fevers and worsening abdominal pain of 5 weeks duration. She had a history of travel to a less developed country and exposure to possible infectious diseases. Abdominal imaging and blood tests revealed diffuse mesenteric lymphadenopathy, elevated transaminases, and elevation of inflammatory markers. Gastroesophageal and colon endoscopies revealed gastric ulcers, and the patient was discharged with a presumptive diagnosis of systemic juvenile idiopathic arthritis given the lymphadenopathy seen on imaging, serositis, sacroiliac joint stiffness noted on physical examination, and pain relief with celecoxib. She presented again 4 days later with worsening abdominal tenderness, elevated transaminases, and new-onset abdominal distention. Tissue biopsy yielded the diagnosis and directed appropriate treatment.

A noncanonical role for the engulfment gene ELMO1 in neutrophils that promotes inflammatory arthritis.

Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.

Rethinking Phagocytes: Clues from the Retina and Testes.

Specialized phagocytes are a newly appreciated classification of phagocyte that currently encompasses Sertoli cells (SCs) of the testes and the retinal pigment epithelial cells (RPE) of the retina. While these cells support very different tissues, they have a striking degree of similarity both as phagocytes and in ways that go beyond cell clearance. The clearance of apoptotic germ cells, cell debris, and used photoreceptor outer segments are critical functions of these cells, and the unique nature of their clearance events make specialized phagocytes uniquely suited for studying the larger implications of cell clearance in vivo. The shared functions of specialized phagocytes could provide novel insights into how phagocytosis impacts tissue homeostasis and immune modulation. In this review, we examine the remarkable similarities between SCs and RPE as specialized phagocytes and the physiological effects of cell clearance within a tissue.

Context-dependent compensation among phosphatidylserine-recognition receptors.

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk -/- Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk -/- mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk +/+ and Mertk -/- mice. Prior to the onset of photoreceptor degeneration, Mertk -/- mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

Ex vivo modulation of the Foxo1 phosphorylation state does not lead to dysfunction of T regulatory cells.

Peripheral regulatory CD4+ T cells (Treg cells) prevent maladaptive inflammatory responses to innocuous foreign antigens. Treg cell dysfunction has been linked to many inflammatory diseases, including allergic airway inflammation. Glucocorticoids that are used to treat allergic airway inflammation and asthma are thought to work in part by promoting Treg cell differentiation; patients who are refractory to these drugs have defective induction of anti-inflammatory Treg cells. Previous observations suggest that Treg cells deficient in the transcription factor FoxO1 are pro-inflammatory, and that FoxO1 activity is regulated by its phosphorylation status and nuclear localization. Here, we asked whether altering the phosphorylation state of FoxO1 through modulation of a regulatory phosphatase might affect Treg cell function. In a mouse model of house dust mite-induced allergic airway inflammation, we observed robust recruitment of Treg cells to the lungs and lymph nodes of diseased mice, without an apparent increase in the Treg cytokine interleukin-10 in the airways. Intriguingly, expression of PP2A, a serine/threonine phosphatase linked to the regulation of FoxO1 phosphorylation, was decreased in the mediastinal lymph nodes of HDM-treated mice, mirroring the decreased PP2A expression seen in peripheral blood monocytes of glucocorticoid-resistant asthmatic patients. When we asked whether modulation of PP2A activity alters Treg cell function via treatment with the PP2A inhibitor okadaic acid, we observed increased phosphorylation of FoxO1 and decreased nuclear localization. However, dysregulation of FoxO1 did not impair Treg cell differentiation ex vivo or cause Treg cells to adopt a pro-inflammatory phenotype. Moreover, inhibition of PP2A activity did not affect the suppressive function of Treg cells ex vivo. Collectively, these data suggest that modulation of the phosphorylation state of FoxO1 via PP2A inhibition does not modify Treg cell function ex vivo. Our data also highlight the caveat in using ex vivo assays of Treg cell differentiation and function, in that while these assays are useful, they may not fully recapitulate Treg cell phenotypes that are observed in vivo.

Boosting Apoptotic Cell Clearance by Colonic Epithelial Cells Attenuates Inflammation In Vivo.

Few apoptotic corpses are seen even in tissues with high cellular turnover, leading to the notion that the capacity for engulfment in vivo is vast. Whether corpse clearance can be enhanced in vivo for potential benefit is not known. In a colonic inflammation model, we noted that the expression of the phagocytic receptor Bai1 was progressively downmodulated. Consistent with this, BAI1-deficient mice had more pronounced colitis and lower survival, with many uncleared apoptotic corpses and inflammatory cytokines within the colonic epithelium. When we engineered and tested transgenic mice overexpressing BAI1, these had fewer apoptotic cells, reduced inflammation, and attenuated disease. Boosting BAI1-mediated uptake by intestinal epithelial cells (rather than myeloid cells) was important in attenuating inflammation. A signaling-deficient BAI1 transgene could not provide a similar benefit. Collectively, these complementary genetic approaches showed that cell clearance could be boosted in vivo, with potential to regulate tissue inflammation in specific contexts.

Apoptotic cell recognition receptors and scavenger receptors.

Phosphatidylserine recognition receptors are a highly diverse set of receptors grouped by their ability to recognize the 'eat-me' signal phosphatidylserine on apoptotic cells. Most of the phosphatidylserine recognition receptors dampen inflammation by inducing the production of anti-inflammatory mediators during the phagocytosis of apoptotic corpses. However, many phosphatidylserine receptors are also capable of recognizing other ligands, with some receptors being categorized as scavenger receptors. It is now appreciated that these receptors can elicit different downstream events for particular ligands. Therefore, how phosphatidylserine recognition receptors mediate specific signals during recognition of apoptotic cells versus other ligands, and how this might help regulate the inflammatory state of a tissue is an important question that is not fully understood. Here, we revisit the work on signaling downstream of the phosphatidylserine recognition receptor BAI1, and evaluate how these and other signaling modules mediate signaling downstream from other receptors, including Stabilin-2, MerTK, and αvß5. We also propose the concept that phosphatidylserine recognition receptors could be viewed as a subset of scavenger receptors that are capable of eliciting anti-inflammatory responses to apoptotic cells.

Apoptosis and engulfment by bronchial epithelial cells. Implications for allergic airway inflammation.

Insult or injury to the lung epithelial cells from pathogens, pollutants, and allergens can initiate the process of apoptotic cell death. Although "Creola bodies," which are clusters of uncleared, apoptotic, epithelial cells, have been seen in the sputum of patients with asthma, the clearance of these dying epithelial cells and the consequence of failed clearance in the airway have not been directly addressed. We have observed that bronchial epithelial cells efficiently engulf their apoptotic neighbors and produce antiinflammatory cytokines when engulfing apoptotic cells. Furthermore, when the phagocytic capacity of bronchial epithelial cells was impaired, mice developed severe, IL-33-dependent, allergic airway inflammation. This inflammation could be ameliorated by exogenous administration of the antiinflammatory cytokine IL-10. Our data suggest that the process of apoptotic cell engulfment is a mechanism by which bronchial epithelial cells regulate the inflammatory environment within the lung. Collectively, these studies suggest that impaired engulfment pathways in airway epithelial cells can contribute to allergic airway inflammation and that targeting these pathways may be of benefit in human airway inflammation.

Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

Impaired fitness of Mycobacterium africanum despite secretion of ESAT-6.

BACKGROUND: When compared with Mycobacterium tuberculosis, individuals that live in the same household as an active case of smear-positive pulmonary tuberculosis exposed to M. africanum progress less frequently to active disease within 2 years. A putative ESX-1 secretion apparatus member, Rv3879c, is mutated in M. africanum, and individuals infected with M. africanum less frequently demonstrate T-cell responses to the ESX-1-secreted virulence factor ESAT-6 than those infected with M. tuberculosis. We hypothesized that less frequent progression is caused by impaired secretion of ESAT-6. METHODS: We analyzed in vivo growth and in vitro secretion of ESAT-6 and CFP-10, comparing M. tuberculosis to M. africanum and a strain of M. africanum complemented with M. tuberculosis Rv3879c. RESULTS: ESAT-6 and CFP-10 secretion were similar for all strains, although these were enriched in M. africanum cell lysates, suggesting a modest ESX-1 secretion defect unrelated to the Rv3879c mutation. In mice, M. africanum demonstrated smaller bacterial population sizes than M. tuberculosis but similar numbers and frequencies of ESAT-6-responsive T cells in the lungs. CONCLUSIONS: These results confirm impaired fitness of M. africanum in vivo and indicate that Rv3879c is not required for secretion of ESAT-6 or for its presentation as an antigen to T cells in vivo.