Rapid clearance of adoptively transferred Cd47-null (Cd47^−/−) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein α (SIRPα) triggers inhibitory signaling through SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47^−/− mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirpα-KO (Sirpα^−/−) mice, as well as Cd47^−/− mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRPα axis dominantly controlling macrophage behavior. Sirpα^−/− mice and Cd47^−/− mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirpα^−/− or Cd47^−/− mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1β, and TNFα, but not IFNγ, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47^−/− cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk–mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin. Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47^−/− or Sirpα^−/− mice. Significance The present study reveals that macrophage phagocytosis toward healthy self-cells is controlled by a two-tier mechanism: a forefront activation mechanism requiring the inflammatory cytokine-stimulated protein kinase C (PKC)-spleen tyrosine kinase (Syk) pathway, to which IL-10 conversely regulates, and the subsequent self-target discrimination mechanism controlled by the CD47-signal regulatory protein α (SIRPα)–mediated inhibition. The findings significantly expand our understanding of macrophage phagocytic plasticity and behavior under different conditions and also provide insights into strategies for enhancing transplantation tolerance and macrophage-based cancer eradication, especially for cancers toward which therapeutic antibodies are yet unavailable.

CD47, a self recognition marker expressed on tissue cells, interacts with immunoreceptor SIRPα expressed on the surface of macrophages to initiate inhibitory signaling that prevents macrophage phagocytosis of healthy host cells. Previous studies suggested that cells may lose surface CD47 during aging or apoptosis to enable phagocytic clearance. In the current study, we demonstrate that the level of cell surface CD47 is not decreased, but the distribution pattern of CD47 is altered, during apoptosis. On nonapoptotic cells, CD47 molecules are clustered in lipid rafts forming punctates on the surface, whereas on apoptotic cells, CD47 molecules are diffused on the cell surface following the disassembly of lipid rafts. We show that clustering of CD47 in lipid rafts provides a high binding avidity for cell surface CD47 to ligate macrophage SIRPα, which also presents as clusters, and elicits SIRPα-mediated inhibitory signaling that prevents phagocytosis. In contrast, dispersed CD47 on the apoptotic cell surface is associated with a significant reduction in the binding avidity to SIRPα and a failure to trigger SIRPα signal transduction. Disruption of plasma membrane lipid rafts with methyl-β-cyclodextrin diffuses CD47 clusters, leading to a decrease in the cell binding avidity to SIRPα and a concomitant increase in cells being engulfed by macrophages. Taken together, our study reveals that CD47 normally is clustered in lipid rafts on nonapoptotic cells but is diffused in the plasma membrane when apoptosis occurs; this transformation of CD47 greatly reduces the strength of CD47–SIRPα engagement, resulting in the phagocytosis of apoptotic cells.

Chronic diseases are often associated with altered inflammatory response, leading to increased host vulnerability to new inflammatory challenges. Employing streptozotocin (STZ)-induced diabetes as a model, we further investigate mechanisms leading to enhanced neutrophil (polymorphonuclear leukocytes [PMN]) responses under hyperglycemia and compare them with those under chronic colitis. We show that, different from colitis under which the PMN response is significantly potentiated, the existence of a proinflammatory state associated with broad increases in macrophages in various organs plays a dominant role in promoting the PMN inflammatory response in diabetic mice. Studies of PMN infiltration during zymosan-induced peritonitis reveal that hyperglycemia enhances PMN recruitment not through inducing a high level of IL-17, which is the case in colitis, but through increasing F4/80⁺ macrophages in the peritoneal cavity, resulting in elevations of IL-6, IL-1β, TNF-α, and CXCL1 production. Insulin reversal of hyperglycemia, but not the neutralization of IL-17, reduces peritoneal macrophage numbers and ameliorates PMN infiltration during peritonitis. Significantly increased macrophages are also observed in the liver, kidneys, and intestines under hyperglycemia, and they are attributable to exacerbated nephropathy and colitis when inflammatory conditions are induced by doxorubicin and dextran sulfate sodium, respectively. Furthermore, analyses of monocyte production and macrophage proliferation in tissues suggest that significant monocytosis of inflammatory F4/80⁺Gr-1⁺ monocytes from the spleen and macrophage proliferation in situ synergistically contribute to the increased macrophage population under hyperglycemia. In conclusion, our results demonstrate that STZ-induced hyperglycemic mice develop a systemic proinflammatory state mediated by broad infiltration of macrophages.

Although multiple instance learning (MIL) methods are widely used for automatic tumor detection on whole slide images (WSI), they suffer from the extreme class imbalance WSIs containing small tumors where the tumor may include only a few isolated cells. For early detection, it is important that MIL algorithms can identify small tumors. Existing studies have attempted to address this issue using attention-based architectures and instance selection-based methodologies but have not produced significant improvements. This paper proposes crossattention-based salient instance inference MIL (CASiiMIL), which involves a novel saliency-informed attention mechanism to identify small tumors (e.g., breast cancer lymph node micro-metastasis) on WSIs without needing any annotations. In addition to this new attention mechanism, we introduce a negative representation learning algorithm to facilitate the learning of saliencyinformed attention weights for improved sensitivity on tumor WSIs. The proposed model outperforms the state-ofthe-art MIL methods on two popular tumor metastasis detection datasets. The proposed approach demonstrates great cross-center generalizability, high accuracy in classifying WSIs with small tumor lesions, and excellent interpretability attributed to the saliency-informed attention weights. We expect that the proposed method will pave the way for training algorithms for early tumor detection on large datasets where acquiring fine-grained annotations is is not practical

Retrospective cohort study. Reported incidences and complications of heterotopic ossification (HO) after using recombinant human bone morphogenetic protein–2 (rhBMP-2) in transforaminal lumbar interbody fusion (TLIF) have been inconclusive. This study was designed to evaluate both incidences of radiologic and symptomatic HO in a large series of TLIFs using rhBMP-2. A total of 996 disc levels in 927 consecutive TLIF patients were retrospectively evaluated at 6-month postoperative follow-up in a single surgical practice. Subjects were separated into the BMP group and the control group. Operative reports, pre- and postoperative medical records were reviewed. Computed tomography (CT) scans were analyzed and graded independently for ossification at each disc level of TLIF. A total of 933 disc levels were in the BMP group, and 63 were in the control group. Six-month fusion rate of interbody was 92.5% in the BMP group, which was significantly higher in contrast to 71.4% in the control group (P < .001). The incidence of radiologic HO in the BMP group was 13.5%, which was significantly higher than 1.6% in the control group (P = .006). After controlling for basic demographics and comorbidities, the presence of radiologic HO was significantly associated with the use of rhBMP-2 (P = .026). However, only one case in the BMP group (0.11%) developed a symptomatic HO (mild-medium left buttock pain, treated nonsurgically) involving left foramen of L5-S1. rhBMP-2 can be safely used in TLIF with regard to HO. There was a low rate of radiologic HO and minimal symptomatic HO, with high fusion rates at 6 months postoperative.

Study Design: A retrospective cohort of prospective data. Objective: Determine the frequency of various symptoms in a surgical cohort of cervical myelopathy (CM). Summary of Background Data: CM can be difficult to diagnose as there is no sine qua non “myelopathic symptom.” Despite extensive literature, the likelihood or frequency of symptoms at presentation remains unclear. Materials and Methods: A total of 484 patients treated at a single academic center were reviewed. Preoperative symptoms included: axial neck pain; upper extremity (UE) pain; UE sensory or motor deficit; lower extremity (LE) sensory or motor deficit; and sphincter dysfunction. It was noted whether a symptom was the chief complaint (CC) and/or one of a list of overall symptoms (OS) reported by the patient. Magnetic resonance imaging was assessed for the maximal cord compression level and T2 hyperintensity. Results: The most common CC was UE sensory deficit (46.5%), whereas the most common OS were UE and LE motor deficits (82.6% and 81.2%). Neck pain was significantly less common (32.6% CC, 55.4% OS), and sphincter dysfunction was rare (0.6% CC, 16.5% OS). UE pain as a CC was significantly higher when the maximal compression involved a more distal level. The presence of T2 hyperintensity was negatively associated with neck pain but positively associated with sensory and motor deficits of LE. Conclusions: The most common CC in CM related to UE sensation, whereas the most common OS related to upper and lower motor function. UE pain was more common with more distal cord compression. Those with T2 hyperintensity had worse myelopathy and were less likely to have neck pain, but more likely to have LE symptoms. To our knowledge, this study is the largest to quantify the frequency of myelopathic symptom presentation in a surgical population. These findings provide valuable insight into the symptomatic presentation of CM in clinical practice and can be used to better inform diagnosis and treatment in this complex patient population. Level of Evidence: Level II—retrospective study.