Single-Cell Analysis Reveals a Subset of High IL-12p40-Secreting Dendritic Cells within Mouse Bone Marrow-Derived Macrophages Differentiated with M-CSF.

Macrophages and dendritic cells (DCs), although ontogenetically distinct, have overlapping functions and exhibit substantial cell-to-cell heterogeneity that can complicate their identification and obscure innate immune function. In this study, we report that M-CSF-differentiated murine bone marrow-derived macrophages (BMDMs) exhibit extreme heterogeneity in the production of IL-12, a key proinflammatory cytokine linking innate and adaptive immunity. A microwell secretion assay revealed that a small fraction of BMDMs stimulated with LPS secrete most IL-12p40, and we confirmed that this is due to extremely high expression of Il12b, the gene encoding IL-12p40, in a subset of cells. Using an Il12b-YFP reporter mouse, we isolated cells with high LPS-induced Il12b expression and found that this subset was enriched for genes associated with the DC lineage. Single-cell RNA sequencing data confirmed a DC-like subset that differentiates within BMDM cultures that is transcriptionally distinct but could not be isolated by surface marker expression. Although not readily apparent in the resting state, upon LPS stimulation, this subset exhibited a typical DC-associated activation program that is distinct from LPS-induced stochastic BMDM cell-to-cell heterogeneity. Overall, our findings underscore the difficulty in distinguishing macrophages and DCs even in widely used in vitro murine BMDM cultures and could affect the interpretation of some studies that use BMDMs to explore acute inflammatory responses.

Apoptosis recognition receptors regulate skin tissue repair in mice.

Apoptosis and clearance of apoptotic cells via efferocytosis are evolutionarily conserved processes that drive tissue repair. However, the mechanisms by which recognition and clearance of apoptotic cells regulate repair are not fully understood. Here, we use single-cell RNA sequencing to provide a map of the cellular dynamics during early inflammation in mouse skin wounds. We find that apoptotic pathways and efferocytosis receptors are elevated in fibroblasts and immune cells, including resident Lyve1+ macrophages, during inflammation. Interestingly, human diabetic foot wounds upregulate mRNAs for efferocytosis pathway genes and display altered efferocytosis signaling via the receptor Axl and its ligand Gas6. During early inflammation in mouse wounds, we detect upregulation of Axl in dendritic cells and fibroblasts via TLR3-independent mechanisms. Inhibition studies in vivo in mice reveal that Axl signaling is required for wound repair but is dispensable for efferocytosis. By contrast, inhibition of another efferocytosis receptor, Timd4, in mouse wounds decreases efferocytosis and abrogates wound repair. These data highlight the distinct mechanisms by which apoptotic cell detection coordinates tissue repair and provides potential therapeutic targets for chronic wounds in diabetic patients.

Our skin is constantly exposed to potential damage from the outside world, and it is vital that any injuries are repaired quickly and effectively. Diabetes and many other health conditions can hamper wound healing, resulting in chronic wounds that are both painful and at risk of becoming infected, which can lead to serious illness and death of patients. After an injury to the skin, the wound becomes inflamed as immune cells rush to the site of injury to fight off infection and clear the wound of dead cells and debris. Some of these dead cells will have died by a highly controlled process known as apoptosis. These so-called apoptotic cells display signals on their surface that nearby healthy cells recognize. This triggers the healthy cells to eat the apoptotic cells to remove them from the wound. Previous studies have linked changes in cell death and the removal of dead cells to chronic wounds in patients with diabetes, but it remains unclear how removing dead cells from the wound affects healing. Justynski et al. used a genetic technique called single-cell RNA sequencing to study the patterns of gene activity in mouse skin cells shortly after a wound. The experiments found that, as the area around the wound started to become inflamed, the wounded cells produced signals of apoptosis that in turn triggered nearby healthy cells to remove them. Other signals relating to the removal of dead cells were also widespread in the mouse wounds and treating the wounds with drugs that inhibit these signals resulted in multiple defects in the healing process. Further experiments used the same approach to study samples of tissue taken from foot wounds in human patients with or without diabetes. This revealed that several genes involved in the removal of dead cells were more highly expressed in the wounds of diabetic patients than in the wounds of other individuals. These findings indicate that for wounds to heal properly it is crucial for the body to detect and clear apoptotic cells from the wound site. Further studies building on this work may help to explain why some diabetic patients suffer from chronic wounds and help to develop more effective treatments for them.

Combinatorial Immunotherapy with Agonistic CD40 Activates Dendritic Cells to Express IL12 and Overcomes PD-1 Resistance.

Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1-resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours after therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DC) using a novel microwell assay identified a CCL22+CCL5+ IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance.

Functionally and Metabolically Divergent Melanoma-Associated Macrophages Originate from Common Bone-Marrow Precursors.

Tumor-associated macrophages (TAMs) can be widely heterogeneous, based on their ontogeny and function, and driven by the tissue-specific niche. TAMs are highly abundant in the melanoma tumor microenvironment (TME), usually correlating with worse prognoses. However, the understanding of their diversity may be harnessed for therapeutic purposes. Here, we used the clinically relevant YUMM1.7 model to study melanoma TAM origin and dynamics during tumor progression. In i.d. YUMM1.7 tumors, we identified distinct TAM subsets based on F4/80 expression, with the F4/80high fraction increasing over time and displaying a tissue-resident-like phenotype. While skin-resident macrophages showed mixed ontogeny, F4/80+ TAM subsets in the melanoma TME originated almost exclusively from bone-marrow precursors. A multiparametric analysis of the macrophage phenotype showed a temporal divergence of the F4/80+ TAM subpopulations, which also differed from the skin-resident subsets and their monocytic precursors. Overall, the F4/80+ TAMs displayed co-expressions of M1- and M2-like canonical markers, while RNA sequencing showed differential immunosuppressive and metabolic profiles. Gene-set enrichment analysis (GSEA) revealed F4/80high TAMs to rely on oxidative phosphorylation, with increased proliferation and protein secretion, while F4/80low cells had high pro-inflammatory and intracellular signaling pathways, with lipid and polyamine metabolism. Overall, we provide an in-depth characterization of and compelling evidence for the BM-dependency of melanoma TAMs. Interestingly, the transcriptomic analysis of these BM-derived TAMs matched macrophage subsets with mixed ontogeny, which have been observed in other tumor models. Our findings may serve as a guide for identifying potential ways of targeting specific immunosuppressive TAMs in melanoma.

Functionally and metabolically divergent melanoma-associated macrophages originate from common bone-marrow precursors.

Melanomas display high numbers of tumor-associated macrophages (TAMs), which correlate with worse prognosis. Harnessing macrophages for therapeutic purposes has been particularly challenging due to their heterogeneity, based on their ontogeny and function and driven by the tissue-specific niche. In the present study, we used the YUMM1.7 model to better understand melanoma TAM origin and dynamics during tumor progression, with potential therapeutic implications. We identified distinct TAM subsets based on F4/80 expression, with the F4/80 high fraction increasing over time and displaying tissue-resident-like phenotype. While skin-resident macrophages showed mixed on-togeny, F4/80 + TAM subsets in i.d. YUMM1.7 tumors originated almost exclusively from bone-marrow precursors. Mul-tiparametric analysis of macrophage phenotype showed a temporal divergence of F4/80 + TAM subpopulations, which also differed from skin-resident subsets, and from their monocytic precursors. Overall, F4/80 + TAMs displayed co-ex-pression of M1- and M2-like canonical markers, while RNA-seq and pathway analysis showed differential immunosup-pressive and metabolic profiles. GSEA showed F4/80 high TAMs to rely on oxidative phosphorylation, with increased proliferation and protein secretion while F4/80 low cells had high pro-inflammatory and intracellular signaling pathways, with lipid and polyamine metabolism. Overall, the present in-depth characterization provides further evidence of the ontogeny of the evolving melanoma TAMs, whose gene expression profiles matched recently-identified TAM clusters in other tumor models and human cancers. These findings provide evidence for potentially targeting specific immunosup-pressive TAMs in advanced tumor stages.

Apoptosis recognition receptors regulate skin tissue repair in mice.

Apoptosis and clearance of apoptotic cells via efferocytosis are evolutionarily conserved processes that drive tissue repair. However, the mechanisms by which recognition and clearance of apoptotic cells regulate repair are not fully understood. Here, we use single-cell RNA sequencing to provide a map of the cellular dynamics during early inflammation in mouse skin wounds. We find that apoptotic pathways and efferocytosis receptors are elevated in fibroblasts and immune cells, including resident Lyve1 + macrophages, during inflammation. Interestingly, human diabetic foot wounds upregulate mRNAs for apoptotic genes and display increased and altered efferocytosis signaling via the receptor Axl. During early inflammation in mouse wounds, we detect upregulation of Axl in dendritic cells and fibroblasts via TLR3-independent mechanisms. Inhibition studies in vivo in mice reveal that Axl signaling is required for wound repair but is dispensable for efferocytosis. By contrast, inhibition of another efferocytosis receptor, Timd4, in mouse wounds decreases efferocytosis and abrogates wound repair. These data highlight the distinct mechanisms by which apoptotic cell detection coordinates tissue repair and provides potential therapeutic targets for chronic wounds in diabetic patients.

Langerhans cells are essential components of the angiogenic niche during murine skin repair.

Angiogenesis, the growth of new blood vessels from pre-existing vessels, occurs during development, injury repair, and tumorigenesis to deliver oxygen, immune cells, and nutrients to tissues. Defects in angiogenesis occur in cardiovascular and inflammatory diseases, and chronic, non-healing wounds, yet treatment options are limited. Here, we provide a map of the early angiogenic niche by analyzing single-cell RNA sequencing of mouse skin wound healing. Our data implicate Langerhans cells (LCs), phagocytic, skin-resident immune cells, in driving angiogenesis during skin repair. Using lineage-driven reportersw, three-dimensional (3D) microscopy, and mouse genetics, we show that LCs are situated at the endothelial cell leading edge in mouse skin wounds and are necessary for angiogenesis during repair. These data provide additional future avenues for the control of angiogenesis to treat disease and chronic wounds and extend the function of LCs beyond their canonical role in antigen presentation and T cell immunity.

Mapping and Validation of scRNA-Seq-Derived Cell-Cell Communication Networks in the Tumor Microenvironment.

Recent advances in single-cell technologies, particularly single-cell RNA-sequencing (scRNA-seq), have permitted high throughput transcriptional profiling of a wide variety of biological systems. As scRNA-seq supports inference of cell-cell communication, this technology has and continues to anchor groundbreaking studies into the efficacy and mechanism of novel immunotherapies for cancer treatment. In this review, we will highlight methods developed to infer inter- and intracellular signaling from scRNA-seq and discuss how they have contributed to studies of immunotherapeutic intervention in the tumor microenvironment (TME). However, a central challenge remains in validating the hypothesized cell-cell interactions. Therefore, this review will also cover strategies for integration of these scRNA-seq-derived interaction networks with existing experimental and computational approaches. Integration of these networks with imaging, protein secretion measurements, and network analysis and mathematical modeling tools addresses challenges that remain with scRNA-seq to enhance studies of immunosuppressive and immunotherapy-altered signaling in the TME.

The Critical Need to Recognize That Families Matter for Adult Health: A Systematic Review of the Literature.

A systemic approach to researching families and health should capture the complex network within which family members are embedded, including multiple family relationships and larger systems of health care. However, much of the families and health research focused on adult family members has focused solely on intimate partnerships, usually the marital relationship. This neglects the remainder of the powerfully influencing family relationships adults retain, and may increasingly focus on as they age. We conducted a systematic review of the families and adult health literature, retaining 72 articles which were subsequently thematically coded to highlight main foci of this area of research. Results highlight six themes, which include family relationship quality, family composition, behavioral factors in health and health care, psychophysiological mediators, caregiving, and aging health. Findings support an underrepresentation of family members, other than the intimate partner, in research on adult health.

Un enfoque sistémico de la investigación sobre las familias y la salud debería captar la red compleja dentro de la cual están insertados los familiares, incluidas las relaciones entre varias familias y los sistemas más amplios de asistencia sanitaria. Sin embargo, gran parte de la investigación sobre las familias y la salud centrada en los familiares adultos se ha concentrado únicamente en las relaciones íntimas, generalmente en las relaciones conyugales. Esto desatiende el resto de las relaciones familiares fuertemente influyentes que lo adultos conservan, y en las que posiblemente se centren cada vez más a medida que envejecen. Realizamos un análisis sistemático de la bibliografía sobre las familias y la salud de los adultos, y conservamos 72 artículos que posteriormente se codificaron temáticamente para destacar los ejes principales de esta área de investigación. Los resultados recalcan seis temas, entre los cuales se encuentran: la calidad de las relaciones familiares, la composición familiar, los factores conductuales en la salud y la asistencia sanitaria, los mediadores psicofisiológicos, el cuidado, y la salud en la vejez. Los resultados respaldan una subrepresentación de los familiares aparte de la pareja íntima en las investigaciones sobre la salud de los adultos.

Assessing the Quality of the After-Visit Summary (AVS) in a Primary-Care Clinic.

BACKGROUND AND OBJECTIVE: As part of Affordable Care Act, the Centers for Medicaid Services (CMS) recommend physicians provide patients with an After-Visit Summary (AVS) following a clinic visit. Information should be relevant and actionable with specific instructions regarding their visit and health. Until recently, this recommendation was included as part of meeting the standard for Stage 1 Meaningful Use for all physicians using electronic-health-record (EHR) technology. In 2016, CMS issued a Notice of Proposed Rulemaking to institute parts of the Medicare Access and CHIP Reauthorization Act of 2015 Merit-based Incentive Payment System, which continues to focus on quality, resource use, and use of certified EHR technology. The purpose of this study was to assess the usefulness of the AVS for patients seen at the Parkland Family Medicine Residency Clinic. METHODS: Electronic medical records of 250 randomly selected patients seen at the Parkland Family Medicine Residency Clinic between July 2013 and July 2014 were reviewed using the 3 W's question format, a modified version of the National Patient Safety Foundation's "Ask Me 3 Program," designed to improve communication between patients and their health care providers. RESULTS: The goal of the quality improvement study was to ensure that all patients receive a meaningful (relevant, accurate, and actionable) AVS after each clinic visit. Chart review indicated that 100% of patients received an AVS after each clinic visit. Of these patients, 51.2% were Spanish speaking, 47.2% English speaking, and 1.6% spoke neither English nor Spanish. Of the non-English-speaking patients, 84.8% received the AVS in their first language; the other 15.2% received the AVS in English. Sixteen percent (16%) of patients overall were considered to have received a nonmeaningful AVS. Reasons for the AVS not being meaningful included not containing any information on the patient's presenting problem (39.2%), physician intervention (35%), or plan of care (18.4%). CONCLUSIONS: This study confirmed that although we demonstrate meaningful use of our EHR system, the content of the AVS needs to be improved on.

PedHITSS: A Screening Tool to Detect Childhood Abuse in Clinical Settings.

BACKGROUND AND OBJECTIVES: Though child abuse is prevalent and detrimental, health care providers fail to screen for abuse at sufficient rates to detect or preempt events. Current child abuse screening tools lack brevity and usefulness in clinical settings. To validate the Pediatric Hurt-Insult-Threaten-Scream-Sex (PedHITSS) screening tool, a 5-item questionnaire designed to detect and prompt provider investigation into child abuse in clinical settings, the PedHITSS was compared to the Conflict Tactics Scale: Parent-Child Version (CTSPC) screening measure. METHODS: Participants included 422 pediatric patients (n=242 nonabused; n=180 abused subsample) recruited from an ambulatory care setting, a medical center at-risk referral clinic, or homeless shelter clinic. Parents were asked to complete a cross-sectional survey, including PedHITSS and CTSPC questionnaires. Concurrent validity of PedHITSS was tested with 242 participants identified as nonabused. Construct validity was assessed with 180 participants previously identified as victims of child abuse. RESULTS: Concurrent validity between the CTSPC and PedHITSS was strong, rs=.70 (P<.01). Sensitivity and specificity for correctly identifying abuse victims (≤12 years) was optimal at a cutpoint of one or greater. There was no significant difference in sensitivity and specificity of HITSS and CTSPC in correctly identifying victims of child abuse. CONCLUSIONS: This study indicates that PedHITSS performs as well as CTSPC in identifying and differentiating victims and nonvictims of child abuse. PedHITSS allows health care providers to confidently screen and report suspected cases of child abuse and serves as a mechanism to confirm abuse status through validated means.