Local Proliferation Research Articles

Analysis of the pathogenic characteristics of fungal bloodstream infection in severe burn patients

Objective: To retrospectively analyze the diagnosis time, pathogen distribution, and drug resistance of fungal bloodstream infection in severe burn patients. Methods: Blood samples were collected from 55 severe burn patients with fungal bloodstream infection (including 46 males and 9 females, aged 42 (1, 78) years) admitted to the intensive care unit of the Institute of Burn Research of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) from July 2011 to May 2019 for retrospective analysis. Microbial monitoring system was used to cultivate pathogens, API yeast identification kit and Candida chromogenic medium were used to identify pathogens, and Kirby-Bauer paper disk diffusion method was used to detect drug resistance of fungi to fluconazole, amphotericin B, itraconazole, ketoconazole, and voriconazole. The positive rate of blood fungal culture, mortality rate, distribution of local fungal proliferation sites, the diagnosis time distribution of fungal bloodstream infection, the distribution of fungal species, resistance to commonly-used antifungal drugs, and the use of antibiotics were assessed. The WHONET 5.6 software was applied to analyze the distribution and drug resistance of fungi. Results: (1) Totally 4 839 blood samples were collected during the 9 years, and 122 strains of fungi were isolated, with positive rate of 2.52%. The mortality rate was 14.55% (8 patients) in 55 patients. Catheter fungal proliferation ranked the first among 30 cases of local fungal proliferation. (2) The diagnosis time of fungal bloodstream infection mainly distributed in ≤1 week of hospitalization [32.73% (18/55)]. (3) Among the 55 strains of fungi detected, the detection rate of Candida parapsilosis ranked the first (21.82%, 12 strains), Candida glabrata was the second (18.18%, 10 strains), and Candida tropicalis was tied with Candida albicans in the third place (14.55%, 8 strains). All the detected fungi were sensitive to amphotericin B, and the resistance rates to voriconazole, fluconazole, itraconazole, and ketoconazole were between 4.5% and 9.1%. (4) Droad-spectrum antibiotics were used in all the 55 patients, ≥3 kinds of antibiotics were used in 44 patients, and 37 patients used antibacterial drugs ≥7 days. Conclusions: The diagnosis time of fungal bloodstream infection in the 55 severe burn patients was mainly within 1 week of hospitalization. Candida parapsilosis is the most commonly detected fungal species. Catheter fungal proliferation occurs most commonly among the 30 patients with local fungal proliferation. All the detected fungi were sensitive to amphotericin B, with low drug resistance to voriconazole, fluconazole, itraconazole, and ketoconazole. Broad-spectrum antibiotics were overused in the severe burn patients with fungal bloodstream infection.

Dry Needling as a Treatment Modality for Tendinopathy: a Narrative Review.

Tendinopathy describes a combination of pain, swelling, and impaired performance of the tendon and around structures. There are various treatment options for tendinopathy with unclear efficacy. Dry needling involves inserting needles into the affected tendon, and it is thought to disrupt the chronic degenerative process and encourage localized bleeding and fibroblastic proliferation. The purpose of this review is to review the use of dry needling as a treatment modality for tendinopathy. The effectiveness of dry needling for treatment of tendinopathy has been evaluated in 3 systematic reviews, 7 randomized controlled trials, and 6 cohort studies. The following sites were studied: wrist common extensor origin, patellar tendon, rotator cuff, and tendons around the greater trochanter. There is considerable heterogeneity of the needling techniques, and the studies were inconsistent about the therapy used after the procedure. Most systematic reviews and randomized controlled trials support the effectiveness of tendon needling. There was a statistically significant improvement in the patient-reported symptoms in most studies. Some studies reported an objective improvement assessed by ultrasound. Two studies reported complications. Current research provides initial support for the efficacy of dry needling for tendinopathy treatment. It seems that tendon needling is minimally invasive, safe, and inexpensive, carries a low risk, and represents a promising area of future research. In further high-quality studies, tendon dry needling should be used as an active intervention and compared with appropriate sham interventions. Studies that compare the different protocols of tendon dry needling are also needed.

Defining molecular identity and fates of CNS-border associated macrophages after ischemic stroke in rodents and humans

Central nervous system (CNS)-border associated macrophages (BAMs) maintain their steady-state population during adulthood and are not replaced by circulating monocytes under physiological conditions. Their roles in CNS integrity and functions under pathological conditions remain largely unknown. Until recently, BAMs and microglia could not be unequivocally distinguished due to expression of common macrophage markers. We investigated the transcriptional profiles of immunosorted BAMs from rat sham-operated and ischemic brains using RNA sequencing. We found that BAMs express the distinct transcriptional signature than microglia and infiltrating macrophages. The enrichment of functional groups associated with the cell cycle in CD163+ cells isolated 3 days after the ischemic injury indicates the proliferative capacity of these cells. The increased number of CD163+ cells 3 days post-ischemia was corroborated by flow cytometry and detecting the increased number of CD163+ cells positive for a proliferation marker Ki67 at perivascular spaces. CD163+ cells in the ischemic brains up-regulated many inflammatory genes and parenchymal CD163+ cells expressed iNOS, which indicates acquisition of a pro-inflammatory phenotype. In mice, BAMs typically express CD206 and we found a subset of these cells expressing CD169. Chimeric mice generated by transplanting bone marrow of donor Cx3cr1gfpCCR2rfp mice to wild type hosts showed an increased number of CX3CR1+CD169+ perivascular macrophages 3 days post-ischemia. Furthermore, these cells accumulated in the brain parenchyma and we detected replacement of perivascular macrophages by peripheral monocytic cells in the sub-acute phase of stroke. In line with the animal results, post-mortem brain samples from human ischemic stroke cases showed time-dependent accumulation of CD163+ cells in the ischemic parenchyma. Our findings indicate a unique transcriptional signature of BAMs, their local proliferation and migration of inflammatory BAMs to the brain parenchyma after stroke in animal models and humans.

Wasp Controls Oriented Migration of Endothelial Cells to Achieve Functional Vascular Patterning

Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. Here, using the zebrafish trunk vasculature we show that in future veins endothelial cells proliferate more than in future arteries and migrate preferentially towards neighboring arteries. In future arteries endothelial cells show a biphasic migration profile. During sprouting cells move away from the dorsal aorta, during remodelling cells stop or move towards the feeding aorta. The final morphology of blood vessels is thus established by local proliferation and oriented cell migration to and from neighboring vessels. Additionally, we identify WASp to be essential for this differential migration. Loss of WASp leads to irregular distribution of endothelial cells, substantially enlarged veins and persistent arteriovenous shunting. Mechanistically, we report that WASp drives the assembly of junctional associated actin filaments and is required for junctional expression of PECAM-1. Together, our data identify that functional vascular patterning in the zebrafish trunk utilizes differential cell movement regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.

CD226 deletion improves post-infarction healing via modulating macrophage polarization in mice.

Macrophages are essential for wound repair after myocardial infarction (MI). CD226, a member of immunoglobulin superfamily, is expressed on inflammatory monocytes, however, the role of CD226 in infarct healing and the effect of CD226 on macrophage remain unknown.Methods: Wild type and CD226 knockout (CD226 KO) mice were subjected to permanent coronary ligation. CD226 expression, cardiac function and ventricular remodeling were evaluated. Profile of macrophages, myofibroblasts, angiogenesis and monocytes mobilization were determined.Results: CD226 expression increased in the infarcted heart, with a peak on day 7 after MI. CD226 KO attenuated infarct expansion and improved infarct healing after MI. CD226 deletion resulted in increased F4/80+ CD206+ M2 macrophages and diminished Mac-3+ iNOS+ M1 macrophages accumulation in the infarcted heart, as well as enrichment of α-smooth muscle actin positive myofibroblasts and Ki67+ CD31+ endothelial cells, leading to increased reparative collagen deposition and angiogenesis. Furthermore, CD226 deletion restrained inflammatory monocytes mobilization, as revealed by enhanced retention of Ly6Chi monocytes in the spleen associated with a decrease of Ly6Chi monocytes in the peripheral blood, whereas local proliferation of macrophage in the ischemic heart was not affected by CD226 deficiency. In vitro studies using bone marrow-derived macrophages showed that CD226 deletion potentiated M2 polarization and suppressed M1 polarization.Conclusion: CD226 expression is dramatically increased in the infarcted heart, and CD226 deletion improves post-infarction healing and cardiac function by favoring macrophage polarization towards reparative phenotype. Thus, inhibition of CD226 may represent a novel therapeutic approach to improve wound healing and cardiac function after MI.

The role of macrophages in obesity-associated islet inflammation and β-cell abnormalities.

Chronic, unresolved tissue inflammation is a well-described feature of obesity, type 2 diabetes mellitus (T2DM) and other insulin-resistant states. In this context, adipose tissue andliver inflammation have been particularly well studied; however, abundant evidence demonstrates that inflammatory processes are also activated in pancreatic islets from obese animals and humans with obesity and/or T2DM. In this Review, we focus on the characteristics ofimmune cell-mediated inflammation in islets and the consequences of this with respect to β-cell function. In contrast to type 1 diabetes mellitus, the dominant immune cell type causing inflammation in obese and T2DM islets is the macrophage. The increased macrophage accumulation in T2DM islets primarily arises through local proliferation of resident macrophages, which then provide signals (such as platelet-derived growth factor) that drive β-cell hyperplasia (a classic feature of obesity). In addition, islet macrophages also impair the insulin secretory capacity of β-cells. Through these mechanisms, islet-resident macrophages underlie the inflammatory response in obesity and mechanistically participate in the β-cell hyperplasia and dysfunction thatcharacterizes this insulin-resistant state. These findings point to the possibility of therapeutics that target islet inflammation to elicit beneficial effects on β-cell function andglycaemia.

Proliferation of Ly6C+ monocytes/macrophages contributes to their accumulation in mouse skin wounds.

Monocytes and macrophages (Mo/MΦ) play critical roles in all phases of skin wound healing. The majority of these cells are thought to be recruited from blood Mo; however, the role local proliferation of Mo/MΦ in the wound has not been defined. Therefore, we tested the hypothesis that local proliferation of Mo and/or MΦ contributes to their accumulation during wound healing. Male C57Bl/6 mice (N=4-9/group) were subjected to excisional skin wounding. Proliferating Mo/MΦ (F4/80+Ki67+) were observed in wound cryosections, peaking on day 5 post-wounding. Cell cycle analysis on cells isolated from skin tissue revealed that wounding increased both the number and percentage of inflammatory Ly6C+F4/80lo/- Mo/MΦ in the S/G2/M phases, peaking on day 6 post-wounding. In contrast, more mature Ly6C-F4/80+ cells were found predominantly in the G0 phase with less than 1% cells in S/G2/M phase following injury. In peripheral blood, Mo were very rarely found in the S/G2/M phase, suggesting that the wound environment triggered the Ly6C+F4/80lo/- Mo proliferative response. Furthermore, injury induced several potential regulators of proliferation in wounds, including IL-1β and IL-6, and wound Mo/MΦ expressed surface receptors for these cytokines. However, wound Mo/MΦ proliferation was not altered in IL-1R1 knockout (KO) or IL-6 KO mice. In summary, our findings indicate that proliferation contributes to Mo/MΦ accumulation in wounds and, contrary to findings in other pathophysiologic conditions, Ly6C+/F4/80lo/- Mo/MΦ proliferate during skin wound healing whereas mature Ly6C-F4/80+ MΦ do not.

Inflammatory macrophages switch to CCL17-expressing phenotype and promote peritoneal fibrosis.

Peritoneal fibrosis remains a problem in kidney failure patients treated with peritoneal dialysis. Severe peritoneal fibrosis with encapsulation or encapsulating peritoneal sclerosis is devastating and life-threatening. Although submesothelial fibroblasts as the major precursor of scar-producing myofibroblasts in animal models and M2 macrophage (Mϕ)-derived chemokines in peritoneal effluents of patients before diagnosis of encapsulating peritoneal sclerosis have been identified, attenuation of peritoneal fibrosis is an unmet medical need partly because the mechanism for cross talk between Mϕs and fibroblasts remains unclear. We use a sodium hypochlorite-induced mouse model akin to clinical encapsulated peritoneal sclerosis to study how the peritoneal Mϕs activate fibroblasts and fibrosis. Sodium hypochlorite induces the disappearance of CD11bhigh F4/80high resident Mϕs but accumulation of CD11bint F4/80int inflammatory Mϕs (InfMϕs) through recruiting blood monocytes and activating local cell proliferation. InfMϕs switch to express chemokine (C-C motif) ligand 17 (CCL17), CCL22, and arginase-1 from day 2 after hypochlorite injury. More than 75% of InfMϕs undergo genetic recombination by Csf1r-driven Cre recombinase, providing the possibility to reduce myofibroblasts and fibrosis by diphtheria toxin-induced Mϕ ablation from day 2 after injury. Furthermore, administration of antibody against CCL17 can reduce Mϕs, myofibroblasts, fibrosis, and improve peritoneal function after injury. Mechanistically, CCL17 stimulates migration and collagen production of submesothelial fibroblasts in culture. By breeding mice that are induced to express red fluorescent protein in Mϕs and green fluorescence protein (GFP) in Col1a1-expressing cells, we confirmed that Mϕs do not produce collagen in peritoneum before and after injury. However, small numbers of fibrocytes are found in fibrotic peritoneum of chimeric mice with bone marrow from Col1a1-GFP reporter mice, but they do not contribute to myofibroblasts. These data demonstrate that InfMϕs switch to pro-fibrotic phenotype and activate peritoneal fibroblasts through CCL17 after injury. CCL17 blockade in patients with peritoneal fibrosis may provide a novel therapy. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Disease Model of Atherosclerosis Using Werner Syndrome Patient-Derived Induced Pluripotent Stem Cells Reveals Excessive Inflammatory Response Against Ox-LDL

Atherosclerosis is a major cause of various fatal diseases, such as myocardial infarction, stroke, and ischemic heart failure worldwide. The risk factors of atherosclerosis are well known, including hypertension, diabetes mellitus, dyslipidemia, smoking and obesity. Growing evidence also suggests that aging is the strongest determinant of stroke, which is less common before 40 years old (Soler and Ruiz 2010). Werner syndrome (WS) is a rare human inherited disorder characterized by the appearance of premature aging followed by early onset of aging-associated diseases including atherosclerosis. Myocardial infarction and cancer are being the most common causes of death in patients with WS (Oshima et al. 1993). However, despite being one of the most common causes of death, the etiology of atherosclerosis in WS patients have not been well documented yet. Therefore, the aim of the study was to assess in vitro model of atherosclerosis using WS patient-derived induced pluripotent stem cells (iPSC) to better understand the etiology and pathogenesis of atherosclerosis in WS patients. Atherosclerosis is a chronic inflammatory disease in which vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC) and macrophages (Mφ) play interactive roles in the progression of the disease. To obtain these cells in vitro, we induced VEC, VSMC and Mφ from the progenitor cell populations, KDR+/CD34+/TRA-1-60-, KDR+/CD34-/TRA-1-60- and CD43+/CD34+ cells respectively, derived from iPSCs by the iPS-sac method we developed previously (Takayama et al. 2008, 2010). We found by co-culture with C3H10T1/2 mouse mesenchymal stromal cell line, KDR+/CD34+ and KDR+/CD34- cell populations were able to efficiently induce VEC and VSMC respectively on day 17. Especially, VEC induction efficiency was ~ 4 and ~ 21 fold as compared with the previously established method with collagen IV and laminin-411 respectively (Ohta et al. 2016; Sone et al. 2007; Taura et al. 2009). Furthermore, to induce Mφ, iPS-sac-like structures in culture were maintained for two weeks to get CD43+ hematopoietic cells. CD43+ cells were then cultured for an additional 7 days with a cytokine cocktail on C3H10T1/2 feeder layer. After 3 weeks of initiating culture, ~ 54% of CD11b+/CD33+ Mφ-like cells were obtained. To examine the responses of the induced VEC- and Mφ-like cells in atherosclerotic conditions, we applied oxidized low-density lipoprotein (Ox-LDL) at the concentration of 10, 50 and 100 μg/mL, and performed qRT-PCR analysis. It is well known that inflammatory response is triggered with the uptake of LDL both in VEC and Mφ, thereby contribute to local inflammation, cell necrosis, apoptosis, VSMC proliferation and fibrosis. Accordingly, in Ox-LDL treated iPSC-derived VEC and Mφ, mRNA expression of IL-1β, IL-6 and TNF-α were increased in a dose dependent manner. A critical event in the early stages of atherosclerosis is the internalization of lipid particles by VEC, and then, these lipids particles are oxidized to form Ox-LDL in the endothelium, leading to an increase in inflammatory cell adhesion molecules (ICAM-1 and VCAM-1) on the endothelium. Subsequently, monocytes are then recruited and differentiated into Mφ and start to uptake Ox-LDL particles ultimately forming foam-cells (Wu et al. 2017). Next, we were enthusiastic to compare the effect of Ox-LDL for inflammatory response in WS iPSC-derived VEC and Mφ with healthy iPSC-derived VEC and Mφ. Although no differences were observed in the induction efficiency, cell proliferation and mean fluorescent intensity of Ox-LDL uptake between WS- and healthy-iPSC-derived VEC and Mφ, qRT-PCR analysis revealed that, VEC derived from WS-iPSC shows much higher mRNA expression of cell adhesion molecule gene (ICAM-1; 2.5 fold and VCAM-1; 3.6 fold), inflammatory cytokine gene (IL-6; 12.5 fold and TGF-β; 4.3 fold) and endothelial dysfunction related gene (ET-1; 5.9 fold and PAI-1; 25.2 fold) compared with healthy-iPSC-derived VEC. Consequently, our results suggest that the higher risk of atherosclerosis in WS patients might be due to excessive inflammation response against Ox-LDL in WS-iPSC-derived cells, which is independent of other risk factors frequently observed in WS, such as diabetes and hyperlipidemia, and might be implicated to understand the pathogenesis and therapeutic strategy against WS and general atherosclerosis. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

CD4+ T Cells Sustain Aggressive Chronic Lymphocytic Leukemia through a CD40L-Independent Mechanism

In Chronic Lymphocytic Leukemia (CLL), mature CD5+ B cells accumulate in lymphoid organs such as bone marrow and lymph nodes where they proliferate and expand within localized proliferation centers. In vitro and in vivo data suggest that survival and proliferation of CLL cells within proliferation centers may be also dependent on microenvironmental interactions originating from the surrounding cellular elements (e.g. monocyte-derived nurse-like cells, mesenchymal stromal cells, or CD4+ T lymphocytes), that deliver both membrane-bound and soluble signals to CLL cells. In particular, the role of CD4+ T cells in vivo is less defined and data in animal models are conflicting as they appear to sustain CLL clone expansion and survival through CD40L-CD40 interactions, though in approximately 40% of patients with CLL, aggressive leukemic clones appear to be independent of CD40 stimulation. We aimed at clarifying the role of CD4+ T lymphocytes taking advantage of the Eμ-TCL1 mouse model, which develops a disease that mimics aggressive, human CLL. To this aim, we generated genetically modified Eμ-TCL1 mice lacking either CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-). In these mice, disease appearance and progression were monitored in lymphoid organs and blood by flow cytometry and immunohistochemistry analyses. Findings were confirmed by adoptive transfer of leukemic clones into mice either lacking CD4+ T cells, or CD40L, or treated with monoclonal antibodies depleting selected T cell populations, or blocking CD40L-CD40 interactions. Interestingly, we observed that CLL clones did not expand in mice either lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development in Eμ-TCL1 mice. On the contrary, in TCL1+/+TAP-/- mice, lacking CD8+ T cells, disease progression was accelerated, suggesting an anti-tumor activity exerted by this subset of T cells. Specificity of CD4+ T cells was marginal for CLL development, as leukemic clones developed regularly in transgenic mice whose CD4+ T cells had TCR with CLL-unrelated specificities. Similarly, TCL1+/+CD40-/- mice developed frank CLL with no differences compared to controls, as well as leukemic clones expanded when transferred into wild type mice treated with monoclonal antibodies blocking CD40, or into CD40L-/- mice, suggesting a dispensable role for CD40/CD40L stimulation in the development of murine CLL. Analysis of peritoneal fluid, spleen, lymph nodes and bone marrow showed similar CLL development in Eμ-TCL1 and TCL1+/+CD40-/- mice. In conclusion, our data demonstrates that CD8+ and CD4+ T cells exert opposite roles in CLL: CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the expansion of CLL clones in Eμ-TCL1 mice through CD40-indipendent, and apparently non-cognate mechanisms. Further studies are warranted to dissect the nature of the molecules, either soluble or membrane-bound, responsible for the interactions occurring between CD4+ T cells and CLL B cells fueling the onset and expansion of CLL cells. Disclosures Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy.

IMMU-01. MYELOID-DERIVED SUPPRESSOR CELL HETEROGENEITY DRIVES GLIOBLASTOMA PROGRESSION IN A SEX-DEPENDENT MANNER

Abstract An immunosuppressive tumor microenvironment is a major factor facilitating glioblastoma (GBM) progression and therapeutic resistance. Immunotherapies have had variable success in improving the outcome of GBM patients, suggesting that there is a need to gain insight into the mechanisms of immunosuppression. Our group previously demonstrated that myeloid-derived suppressor cells (MDSCs) expand in GBM patients and infiltrate tumors, where they suppress the activity of cytotoxic cells. However, the mechanisms by which individual MDSC subsets promote tumorigenesis remain understudied. Using the syngeneic mouse glioma models GL261, CT-2A and SB28, we show that monocytic MDSCs (mMDSCs) are prevalent in tumors and that their frequency is significantly higher in males, who constitute 60% of GBM patients and have a worse prognosis than females. mMDSC abundance was further associated with poor survival, and male mice reached morbidity endpoint earlier. Consistent with preclinical observations, male GBM patient specimens had significantly more IBA+CD204+ immunosuppressive myeloid cells compared to female GBM tissue. In contrast, female tumor-bearing mice had a two-fold increase in circulating granulocytic MDSC (gMDSC) frequency, while this population remained unchanged in males. Female-to-male bone marrow chimeras demonstrated that intrinsic discrepancies in immune cell characteristics drive the sex differences in survival. Consistent with the differential MDSC accumulation pattern, targeting gMDSCs with anti-Ly6G neutralizing antibodies extended the lifespan of female mice without providing a survival advantage to males. However, mMDSCs were protected from the anti-Ly6C depletion strategy due to their systemic and local proliferation, as indicated by ex vivo Ki-67 staining and subsequently confirmed by gene expression analysis. Drug-prediction algorithms using the differential RNA sequencing profiles demonstrated that mMDSCs can be targeted by chemotherapeutics, while immunomodulatory drugs are effective against gMDSCs. Collectively, these findings indicate that MDSC subset variation might represent a therapeutic opportunity for improved therapeutic efficacy of immunotherapies while accounting for sex as a biological variable.

IL-10 secreting B cells regulate periodontal immune response during periodontitis.

Periodontitis is a disease caused by periodontopathogens and is characterized by periodontal inflammation and alveolar bone resorption. As has been proven, host immune responses incite the development and progression of periodontitis. The present study sought to establish B10 cell functions and mechanisms in regulating host immunity during periodontitis. Periodontopathogen-specific B10 cells were purified and then injected into recipients to create the adoptive transfer models. We compared inflammatory cytokines and regulatory T (Treg)/Th17 cell expression in a healthy, normal model, an experimental periodontitis model, and experimental periodontitis model adoptively transferred with B10 cells. Compared with experimental periodontitis animals, our results showed that transfer of B10 cells alleviated alveolar bone resorption (P < 0.05) by reducing periodontal osteoclastogenesis (P < 0.05). Additionally, we found that B10 cell transfer into the experimental periodontitis ones resulted in increased IL-10 (P < 0.05), but decreased IL-17 (P < 0.05) and receptor activator for nuclear factor κB ligand (RANKL) (P < 0.05) gene and protein expression in local lesions. Moreover, adoptive transfer of B10 cells reduced the proportion of Th17 cells (P < 0.05) in the gingiva. The results of our study confirmed that B10 cells can modulate local host immune responses and prevent inflammatory damage of alveolar bone by reducing pro-inflammatory cytokine expression and decreasing local proliferation of Th17 cells.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due toenhanced bacterial clearance, which was causedby IFNɣ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

The Central Nervous System Contains ILC1s That Differ From NK Cells in the Response to Inflammation.

Innate lymphoid cells (ILCs) are tissue resident cells with organ-specific properties. Here, we show that the central nervous system (CNS) encompasses ILCs. In particular, CD3−NK1.1+ cells present in the murine CNS comprise natural killer (NK) cells, ILC1s, intermediate ILC1s (intILC1s) and ex-ILC3s. We investigated the properties of CNS-ILC1s in comparison with CNS-NK cells during steady state and experimental autoimmune encephalomyelitis (EAE). ILC1s characteristically express CXCR3, CXCR6, DNAM-1, TRAIL, and CD200R and display heightened TNF-α production upon stimulation. In addition, ILC1s express perforin and are able to degranulate, although in a lesser extent than NK cells. Within the CNS compartments, ILC1s are enriched in the choroid plexus where very few NK cells are present, and also reside in the brain parenchyma and meninges. During EAE, ILC1s maintain stable IFN-γ and TNF-α levels while in NK cells the production of these cytokines increases as EAE progresses. Moreover, the amount of ILC1s and intILC1s increase in the parenchyma during EAE, but in contrast to NK cells, they show no signs of local proliferation. The upregulation in the inflamed brain of chemokines involved in ILC1 migration, such as CXCL9, CXCL10, and CXCL16 may lead to a recruitment of ILC1s from meninges or choroid plexus into the brain parenchyma. In sum, CNS-ILC1 phenotype, distribution and moderate inflammatory response during EAE suggest that they may act as gatekeepers involved in the control of neuroinflammation.

Voltage-gated sodium channel inhibitor reduces atherosclerosis by modulating monocyte/macrophage subsets and suppressing macrophage proliferation

Inflammatory monocyte and macrophage subset accumulation during the inflammatory response that drives atherosclerosis can exacerbate the extent of atherosclerosis. It has been demonstrated that voltage-gated sodium channels (VGSCs) can regulate cell bioactivities in monocytes/macrophages. We hypothesized that blockade of mononuclear phagocyte VGSCs was atheroprotective through monocyte/macrophage subset modulation and macrophage proliferation suppression in atherosclerotic lesions. In this experimental study, when VGSCs were knocked down with RNA interference plasmid transfection in mouse peripheral blood monocytes and monocyte-macrophage lineage RAW264.7 cells in vitro, the biological characteristics of proliferation, phagocytosis, and migration in RAW264.7 cells declined. In addition, suppression of LPS-induced M1 polarization and facilitation of IL-4-induced M2 polarization were also observed. In an in vivo study, ApoE knockout (ApoE−/−) mice were fed a standard chow diet (CD) or a western diet (WD). After feeding with phenytoin (PHT), no significant differences were detected in plasma lipids, and the anti-inflammatory phenotypes of both monocytes and macrophages were elevated and proinflammatory phenotypes declined. The local proliferation of macrophages was also distinctly suppressed, along with a significant reduction in atheromatous plaques. In conclusion, blockade of VGSCs in the mononuclear phagocyte system reduced atherosclerotic lesions, which may occur through altering monocyte/macrophage subsets and suppressing macrophage proliferation in atherosclerotic plaques. Blockage of VGSCs may play an important role in cardiovascular protection.

Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming

SummaryCD8+ T cell responses to hepatotropic viruses like HBV range from dysfunction to differentiation into effector cells, but the mechanisms underlying these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells –not natural targets of HBV – leads to differentiation into effector cells that form dense, extravascular clusters of rather immotile cells scattered throughout the liver. By contrast, priming by hepatocytes – natural targets of HBV - leads to local activation and proliferation but lack of differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses unveil unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by anti-PD-L1 treatment, but instead respond to IL-2. These findings suggest new immunotherapeutic strategies against chronic HBV infection.

P733Cholesterol uptake triggers macrophage proliferation in the plaque

Abstract Background Guidelines recommend cholesterol lowering for primary and secondary prevention of cardiovascular disease. While lipid lowering has been reported to induce plaque regression, the underlying mechanisms have remained speculative. Purpose We hypothesize that lipid uptake triggers local macrophage proliferation in the plaque, and conversely, statin treatment inhibits local macrophage proliferation leading to plaque regression. Methods Mixed bone marrow chimeras were generated in LDLR−/− mice reconstituted with wild type and scavenger receptor deficient or cholesterol exporter deficient bone marrow cells to study cell autonomous effects on macrophage proliferation. APOE*3-Leiden.huCETP mice with established atherosclerosis were randomized to three groups: Continued cholesterol diet, cholesterol diet supplemented with 0.01% atorvastatin, and cholesterol free diet for 4 weeks to study mechanisms of plaque regression. Results Proliferation of scavenger receptor A and CD36 deficient macrophages with impaired lipid uptake was reduced by 30–50% in the plaque, while ABCA1/ABCG1 exporter deficiency resulted in cholesterol overloading and apoptosis. Oral atorvastatin treatment decreased total plasma cholesterol levels by 50% to the same extend as cholesterol free diet feeding in APOE*3-Leiden.huCETP. Cholesterol lowering resulted in a 50% reduction in local macrophage proliferation and plaque regression with reduced macrophage and lipid contents and increased collagen. GFP bone marrow reconstitution of APOE*3-Leiden.huCETP mice in which the aortas were shielded from irradiation showed infiltrating monocytes to contribute only 11% to the plaque macrophage pool during plaque progression, thereby underscoring the relevance of targeting macrophage proliferation for plaque regression. Finally, rates of macrophage proliferation in human carotid artery plaques correlated with serum LDL-cholesterol levels, in line with our experimental studies. Conclusion Foam cell formation in atherosclerotic plaques triggers their proliferation. Targeting macrophage proliferation leads to plaque regression.

Quantitative ultrasound to better evaluate heterogeneous tumor response to therapy

Heterogenity of tumor structure and function (e.g., stromal density, vascularization, and oxygen distribution) can influence local cellular proliferation and therapeutic resistance. Emerging ultrasound imaging techniques can map parameters related to these features but such approaches are not yet in standard clinical practice. The goal of our work is to develop robust protocols to provide more complete information during therapy by quantitatively mapping and interpreting tumor heterogeneity using emerging ultrasonic techniques. Within murine tumor models Shear Wave Elastography (SWE), Contrast-Enhanced Ultrasound (CEUS) and Quantitative Ultrasound (QUS) techniques have been applied with clinical imaging systems to compare changes observed during anti-angiogenic and cytotoxic therapy as compared to controls. In a Lewis Lung Carcinoma model, mice receiving an anti-angiogenic therapy had the highest levels of necrosis and fibrosis and presented the highest number of correlations between quantitative parameters related to microvascular function and tumor structure. This indirectly demonstrates, for this tumor-type, that quantitative parameters measured with clinical imaging systems can help differentiate tumor therapeutic responses. Promising results with ultrasound localization microscopy and photoacoustics will also be reviewed because of the important new information they offer. Finally, the path towards integration of these quantitative ultrasound techniques in clinical practice will be discussed.

Effects of neighbour location and nutrient distributions on root foraging behaviour of the common sunflower.

Plants regularly encounter patchily distributed soil nutrients. A common foraging response is to proliferate roots within high-quality patches. The influence of the social environment on this behaviour has been given limited attention, despite important fitness consequences of competition for soil resources among plants. Using the common sunflower (Helianthus annuus L.), we compared localized root proliferation in a high-quality patch by plants grown alone to that of plants in two different social environments: with a neighbouring plant sharing equal access to the high-quality patch, and with a neighbouring plant present but farther from the high-quality patch such that the focal individual was in closer proximity to the high-quality patch. Sunflowers grown alone proliferated more roots within high-nutrient patches than lower-nutrient soil. Plants decreased root proliferation within a high-nutrient patch when it was equidistant to a neighbour. Conversely, plants increased root proliferation when they were in closer proximity to the patch relative to a nearby neighbour. Such contingent responses may allow sunflowers to avoid competition in highly contested patches, but to also pre-empt soil resources from neighbours when they have better access to a high-quality patch. We also compared patch occupancy by sunflowers grown alone with two equidistant high-quality patches to occupancy by sunflowers grown with two high-quality patches and a neighbour. Plants grown with a neighbour decreased root length within shared patches but did not increase root length within high-quality patches they were in closer proximity to, perhaps because resource pre-emption may be less important for individuals when resources are more abundant. These results show that nutrient foraging responses in plants can be socially contingent, and that plants may account for the possibility of pre-empting limited resources in their foraging decisions.

Chronic Inflammation Directs an Olfactory Stem Cell Functional Switch from Neuroregeneration to Immune Defense.

Although olfactory mucosa possesses long-lived horizontal basal stem cells (HBCs) and remarkable regenerative capacity, the function of human olfactory neuroepithelium is significantly impaired in chronic inflammatory rhinosinusitis. Here, we show that, while inflammation initially damages olfactory neurons and activates HBC-mediated regeneration, continued inflammation locks HBCs in an undifferentiated state. Global gene expression in mouse HBCs reveals broad upregulation of NF-κB-regulated cytokines and chemokines including CCL19, CCL20, and CXCL10, accompanied by enhancement of "stemness"-related transcription factors. Loss-of-function studies identify an NF-κB-dependent role of HBCs in amplifying inflammatory signaling, contributing to macrophage and Tcell local proliferation. Chronically activated HBCs signal macrophages to maintain immune defense and prevent Treg development. In diseased human olfactory tissue, activated HBCs in a P63+ undifferentiated state similarly contribute to inflammation through chemokine production. These observations establish a mechanism of chronic rhinosinusitis-associated olfactory loss, caused by a functional switch of neuroepithelial stem cells from regeneration to immune defense.