An endogenous inhibitor of angiogenesis downregulated by hypoxia in human aortic valve stenosis promotes disease pathogenesis.

Aortic valve stenosis is the most common valve disease in the western world. Central to the pathogenesis of this disease is the growth of new blood vessels (angiogenesis) within the aortic valve allowing infiltration of immune cells and development of intra-valve inflammation. Identifying the cellular mediators involved in this angiogenesis is important as this may reveal new therapeutic targets which could ultimately prevent the progression of aortic valve stenosis. Aortic valves from patients undergoing surgery for aortic valve replacement or dilation of the aortic arch were examined both ex vivo and in vitro. We now demonstrate that the anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), a non-signalling soluble receptor for vascular endothelial growth factor, is constitutively expressed in non-diseased valves. sFlt-1 expression was, however, significantly reduced in aortic valve tissue from patients with aortic valve stenosis while protein markers of hypoxia were simultaneously increased. Exposure of primary-cultured valve interstitial cells to hypoxia resulted in a decrease in the expression of sFlt-1. We further reveal using a bioassay that siRNA knock-down of sFlt1 in valve interstitial cells directly results in a pro-angiogenic environment. Finally, incubation of aortic valves with sphingosine 1-phosphate, a bioactive lipid-mediator, increased sFlt-1 expression and inhibited angiogenesis within valve tissue. In conclusion, this study demonstrates that sFlt1 expression is directly correlated with angiogenesis in aortic valves and the observed decrease in sFlt-1 expression in aortic valve stenosis could increase valve inflammation, promoting disease progression. This could be a viable therapeutic target in treating this disease.

Induction of experimental diabetes and diabetic nephropathy using anomer-equilibrated streptozotocin in male C57Bl/6J mice.

Streptozotocin (STZ) is widely used to induce experimental diabetes in murine models. However, the ability to induce diabetic nephropathy (DN) is more challenging. It has been recommended to inject STZ at multiple low doses within 15 min after dissolution due to its alleged instability. However, some studies suggest that STZ is stable for days due to equilibration of its two anomers (α and ß), 90 min after dissolution, and that this anomer-equilibrated STZ leads to higher survival rates and persistent hyperglycaemia with minimal weight loss. The aim of this study was to determine an optimal dose of anomer-equilibrated STZ to induce kidney tubular damage and compare it with the more commonly used freshly prepared STZ. We hypothesised that anomer-equilibrated STZ provides a better, reproducible experimental model of diabetes-induced kidney damage with improved animal welfare. Body measurements, fasting glycaemia, insulinemia and renal histology were assessed in male C57Bl/6J at two and six months of age treated with fresh (50 mg/kg) or anomer-equilibrated (dose ranging 35-50 mg/kg) STZ or vehicle control. We demonstrated a dose-dependent effect of anomer-equilibrated STZ on the induction of hypo-insulinaemia and hyperglycaemia, as well as body weight in two-month-old mice. Interestingly, in six-month-old mice STZ leads to body weight loss, independently of STZ preparation mode. Anomer-equilibrated STZ provoked moderate to severe kidney tubule structural damage, resulting in significant kidney hypertrophy, whereas freshly prepared STZ only caused mild alterations. In conclusion, our study proposes that anomer-equilibrated STZ provides a robust murine model of diabetes and early-stage diabetic nephropathy, which can be used to test therapeutic approaches to treat and/or prevent renal damage.

Myeloid PTP1B deficiency protects against atherosclerosis by improving cholesterol homeostasis through an AMPK-dependent mechanism.

OBJECTIVE: Atherosclerosis is a chronic inflammatory process induced by the influx and entrapment of excess lipoproteins into the intima media of arteries. Previously, our lab demonstrated that systemic PTP1B inhibition protects against atherosclerosis in preclinical LDLR-/- models. Similarly, it was shown that myeloid-specific PTP1B ablation decreases plaque formation and ameliorates dyslipidaemia in the ApoE-/- model of atherosclerosis. We hypothesized that the relevant improvements in dyslipidaemia following modification of PTP1B activation may either result from changes in hepatic cholesterol biosynthesis and/or increased uptake and degradation by liver-resident macrophages. We examined this in animal models and patients with coronary artery disease. METHODS: In this study, we determined the cholesterol-lowering effect of myeloid-PTP1B deletion in mice fed a high-fat high-cholesterol diet and examined effects on total cholesterol levels and lipoprotein profiles. We also determined the effects of PTP1B inhibition to oxLDL-C challenge on foam cell formation and cholesterol efflux in human monocytes/macrophages. RESULTS: We present evidence that myeloid-PTP1B deficiency significantly increases the affinity of Kupffer cells for ApoB containing lipoproteins, in an IL10-dependent manner. We also demonstrate that PTP1B inhibitor, MSI-1436, treatment decreased foam cell formation in Thp1-derived macrophages and increased macrophage cholesterol efflux to HDL in an AMPK-dependent manner. We present evidence of three novel and distinct mechanisms regulated by PTP1B: an increase in cholesterol efflux from foam cells, decreased uptake of lipoproteins into intra-lesion macrophages in vitro and a decrease of circulating LDL-C and VLDL-C in vivo. CONCLUSIONS: Overall, these results suggest that myeloid-PTP1B inhibition has atheroprotective effects through improved cholesterol handling in atherosclerotic lesions, as well as increased reverse cholesterol transport. Trial registration Research registry, researchregistry 3235. Registered 07 November 2017, https://www.researchregistry.com/browse-the-registry#home/registrationdetails/5a01d0fce7e1904e93e0aac5/ .

The intracellular signaling pathways governing macrophage activation and function in human atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and plaque formation in arterial vessel walls. Atherosclerotic plaques narrow the arterial lumen to increase the risk of heart attacks, ischemic stroke and peripheral vascular disease, which are major and worldwide health and economic burdens. Macrophage accumulation within plaques is characteristic of all stages of atherosclerosis and their presence is a potential marker of disease activity and plaque stability. Macrophages engulf lipids and modified lipoproteins to form foam cells that express pro-inflammatory and chemotactic effector molecules, stress inducing factors and reactive oxygen species. They control plaque stability and rupture through secretion of metalloproteinases and extracellular matrix degradation. Although macrophages can worsen disease by propagating inflammation, they can stabilize atherosclerotic plaques through tissue remodeling, promoting the formation of a fibrous cap, clearing apoptotic cells to prevent necrotic core formation and through vascular repair. In atherosclerosis, macrophages respond to dyslipidaemia, cytokines, dying cells, metabolic factors, lipids, physical stimuli and epigenetic factors and exhibit heterogeneity in their activation depending on the stimuli they receive. Understanding these signals and the pathways driving macrophage function within developing and established plaques and how they can be pharmacologically modulated, represents a strategy for the prevention and treatment of atherosclerosis. This review focusses on the current understanding of factors controlling macrophage heterogeneity and function in atherosclerosis. Particular attention is given to the macrophage intracellular signaling pathways and transcription factors activated by biochemical and biophysical stimuli within plaques, and how they are integrated to regulate plaque formation and stability.

Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds.

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150-500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

Monocytes Expose Factor XIII-A and Stabilize Thrombi against Fibrinolytic Degradation.

Factor XIII (FXIII) is a transglutaminase that promotes thrombus stability by cross-linking fibrin. The cellular form, a homodimer of the A subunits, denoted FXIII-A, lacks a classical signal peptide for its release; however, we have shown that it is exposed on activated platelets. Here we addressed whether monocytes expose intracellular FXIII-A in response to stimuli. Using flow cytometry, we demonstrate that FXIII-A antigen and activity are up-regulated on human monocytes in response to stimulation by IL-4 and IL-10. Higher basal levels of the FXIII-A antigen were noted on the membrane of the monocytic cell line THP-1, but activity was significantly enhanced following stimulation with IL-4 and IL-10. In contrast, treatment with lipopolysaccharide did not upregulate exposure of FXIII-A in THP-1 cells. Quantification of the FXIII-A activity revealed a significant increase in THP-1 cells in total cell lysates following stimulation with IL-4 and IL-10. Following fractionation, the largest pool of FXIII-A was membrane associated. Monocytes were actively incorporated into the fibrin mesh of model thrombi. We found that stimulation of monocytes and THP-1 cells with IL-4 and IL-10 stabilized FXIII-depleted thrombi against fibrinolytic degradation, via a transglutaminase-dependent mechanism. Our data suggest that monocyte-derived FXIII-A externalized in response to stimuli participates in thrombus stabilization.

Myocardial and Systemic Inflammation in Acute Stress-Induced (Takotsubo) Cardiomyopathy.

BACKGROUND: Acute stress-induced (takotsubo) cardiomyopathy can result in a heart failure phenotype with a prognosis comparable with that of myocardial infarction. In this study, we hypothesized that inflammation is central to the pathophysiology and natural history of takotsubo cardiomyopathy. METHODS: In a multicenter study, we prospectively recruited 55 patients with takotsubo cardiomyopathy and 51 age-, sex-, and comorbidity-matched control subjects. During the index event and at the 5-month follow-up, patients with takotsubo cardiomyopathy underwent multiparametric cardiac magnetic resonance imaging, including ultrasmall superparamagnetic particles of iron oxide (USPIO) enhancement for detection of inflammatory macrophages in the myocardium. Blood monocyte subpopulations and serum cytokines were assessed as measures of systemic inflammation. Matched control subjects underwent investigation at a single time point. RESULTS: Subjects were predominantly middle-aged (64±14 years) women (90%). Compared with control subjects, patients with takotsubo cardiomyopathy had greater USPIO enhancement (expressed as the difference between pre-USPIO and post-USPIO T2*) in both ballooning (14.3±0.6 milliseconds versus 10.5±0.9 milliseconds; P<0.001) and nonballooning (12.9±0.6 milliseconds versus 10.5±0.9 milliseconds; P=0.02) left ventricular myocardial segments. Serum interleukin-6 (23.1±4.5 pg/mL versus 6.5±5.8 pg/mL; P<0.001) and chemokine (C-X-C motif) ligand 1 (1903±168 pg/mL versus 1272±177 pg/mL; P=0.01) concentrations and classic CD14++CD16- monocytes (90±0.5% versus 87±0.9%; P=0.01) were also increased whereas intermediate CD14++CD16+ (5.4±0.3% versus 6.9±0.6%; P=0.01) and nonclassic CD14+CD16++ (2.7±0.3% versus 4.2±0.5%; P=0.006) monocytes were reduced in patients with takotsubo cardiomyopathy. At 5 months, USPIO enhancement was no longer detectable in the left ventricular myocardium, although persistent elevations in serum interleukin-6 concentrations ( P=0.009) and reductions in intermediate CD14++CD16+ monocytes (5.6±0.4% versus 6.9±0.6%; P=0.01) remained. CONCLUSIONS: We demonstrate for the first time that takotsubo cardiomyopathy is characterized by a myocardial macrophage inflammatory infiltrate, changes in the distribution of monocyte subsets, and an increase in systemic proinflammatory cytokines. Many of these changes persisted for at least 5 months, suggesting a low-grade chronic inflammatory state. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT02897739.

Exteriorisation of Mannoses on Human Erythrocyte Membrane Skeleton Provides 'Eat Me' Signals for Oxidatively Damaged Cells to be Cleared By Macrophages: A Pathway Mediating Hemolysis in Sickle Cell Disease.

DISCLOSURES: Vickers: University of Aberdeen: Patents & Royalties: About to apply for patent. Barker: University of Aberdeen: Employment, Patents & Royalties: About to apply for patent. Cao: University of Aberdeen: Patents & Royalties: About to apply for patent.

Similarities and differences in surface receptor expression by THP-1 monocytes and differentiated macrophages polarized using seven different conditioning regimens.

Macrophages are key in orchestrating immune responses to micro-environmental stimuli, sensed by a complex set of surface receptors. The human cell line THP-1 has a monocytic phenotype, including the ability to differentiate into macrophages, providing a tractable, standardised surrogate for human monocyte-derived macrophages. Here we assessed the expression of 49 surface markers including Fc, complement, C-type lectin and scavenger receptors; TIMs; Siglecs; and co-stimulatory molecules by flow cytometry on both THP-1 monocytes and macrophages and following macrophage activation with seven standard conditioning/polarizing stimuli. Of the 34 surface markers detected on macrophages, 18 altered expression levels on activation. From these, expression of 9 surface markers were consistently altered by all conditioning regimens, while 9 were specific to individual polarizing stimuli. This study provides a resource for the study of macrophages and highlights that macrophage polarization states share much in common and the differences do not easily fit a simple classification system.

Pharmacological inhibition of protein tyrosine phosphatase 1B protects against atherosclerotic plaque formation in the LDLR-/- mouse model of atherosclerosis.

Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with type 1 or type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance, due to impaired insulin receptor (IR) signalling. Here, we demonstrate that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR prevents and reverses atherosclerotic plaque formation in an LDLR-/- mouse model of atherosclerosis. Acute (single dose) or chronic PTP1B inhibitor (trodusquemine) treatment of LDLR-/- mice decreased weight gain and adiposity, improved glucose homeostasis and attenuated atherosclerotic plaque formation. This was accompanied by a reduction in both, circulating total cholesterol and triglycerides, a decrease in aortic monocyte chemoattractant protein-1 (MCP-1) expression levels and hyperphosphorylation of aortic Akt/PKB and AMPKα. Our findings are the first to demonstrate that PTP1B inhibitors could be used in prevention and reversal of atherosclerosis development and reduction in CVD risk.

Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice.

BACKGROUND: Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown. PURPOSE: Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal. RESULTS: We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro. CONCLUSIONS: These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.

A critical role for suppressor of cytokine signalling 3 in promoting M1 macrophage activation and function in vitro and in vivo.

Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3(hi) -expressing, but not SOCS1(hi) -expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1ß, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide.

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

Modulation of macrophages by biophysical cues in health and beyond.

Macrophages play a key role in tissue development and homeostasis, innate immune defence against microbes or tumours, and restoring homeostasis through tissue regeneration following infection or injury. The ability to adopt such diverse functions is due to their heterogeneous nature, which is driven largely by their developmental origin and their response to signals they encounter from the microenvironment. The most well-characterized signals driving macrophage phenotype and function are biochemical and metabolic. However, the way macrophages sense and respond to their extracellular biophysical environment is becoming increasingly recognized in the field of mechano-immunology. These biophysical cues can be signals from tissue components, such as the composition and charge of extracellular matrix or topography, elasticity, and stiffness of the tissue surrounding cells; and mechanical forces such as shear stress or stretch. Macrophages are important in determining whether a disease resolves or becomes chronic. Ageing and diseases such as cancer or fibrotic disorders are associated with significant changes in the tissue biophysical environment, and this provides signals that integrate with those from biochemical and metabolic stimuli to ultimately dictate the overall function of macrophages. This review provides a brief overview of macrophage polarization, followed by a selection of commonly recognized physiological and applied biophysical stimuli impacting macrophage activity, and the potential signalling mechanisms driving downstream responses. The effects of biophysical cues on macrophages' function in homeostasis and disease and the associated clinical implications are also highlighted.

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function.

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.

Interleukin-27 Regulates the Function of the Gastrointestinal Epithelial Barrier in a Human Tissue-Derived Organoid Model.

A treatment with direct healing effects on the gastrointestinal epithelial barrier is desirable for inflammatory bowel disease (IBD). Interleukin-27 (IL-27) is an immunoregulatory cytokine, and oral delivery is an effective treatment in murine models of IBD. We aimed to define IL-27 effects on the human gastrointestinal epithelial barrier. We characterised gene and protein expression of permeability mediators in a human colon-derived organoid model. Functional permeability was determined in an organoid-derived 2D monolayer by transepithelial electrical resistance. IL-27 effects on epithelial innate immune responses were assessed through expression of cytokines, anti-microbial peptides and MUC genes. IL-27 effects on wound healing and proliferation were determined in human colon epithelial cell lines. IL-27 led to restoration of permeability regulation following inflammatory cytokine insult (p = 0.001), associated with differential expression of tight junction mediators with decrease in claudin 2 (p = 0.024) and increase in claudin 4 (p < 0.001), E-cadherin (p < 0.001) and zona occludens (p = 0.0014). IL-27 evoked differential gene expression of epithelial-derived innate immune responses (reduced IL1B and IL18, and increased IL33, HBD1, MUC1 and MUC2; p < 0.012). IL-27 induced epithelial barrier wound healing through restitution (p < 0.001), and increased proliferation (p < 0.001) following injury. Overall, IL-27 provokes mucosal healing of the human gastrointestinal epithelial barrier.

A Role for Folate in Microbiome-Linked Control of Autoimmunity.

The microbiome exerts considerable control over immune homeostasis and influences susceptibility to autoimmune and autoinflammatory disease (AD/AID) such as inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1 diabetes (T1D), psoriasis, and uveitis. In part, this is due to direct effects of the microbiome on gastrointestinal (GI) physiology and nutrient transport, but also to indirect effects on immunoregulatory controls, including induction and stabilization of T regulatory cells (T reg). Secreted bacterial metabolites such as short-chain fatty acids (SCFA) are under intense investigation as mediators of these effects. In contrast, folate (vitamin B9), an essential micronutrient, has attracted less attention, possibly because it exerts global physiological effects which are difficult to differentiate from specific effects on the immune system. Here, we review the role of folate in AD/AID with some emphasis on sight-threatening autoimmune uveitis. Since folate is required for the generation and maintenance of T reg , we propose that one mechanism for microbiome-based control of AD/AID is via folate-dependent induction of GI tract T reg , particularly colonic T reg, via anergic T cells (T an). Hence, folate supplementation has potential prophylactic and/or therapeutic benefit in AID/AD.

Reference Intervals for Total T4 and Free T4 in Cynomolgus Macaques (Macaca fascicularis) and Rhesus Macaques (Macaca mulatta).

Thyroid diseases, associated with either increased or decreased concentrations of circulating thyroid hormones, are prevalent in both human and veterinary populations. Hypothyroidism is a differential diagnosis for many medical problems as the disease presents with nonspecific clinical signs that can include lethargy, weight gain, cold intolerance, and dermatologic manifestations such as alopecia. Alopecia is a frequently reported problem in captive nonhuman primates (NHP), and hypothyroidism is considered to be a differential diagnosis. However, thyroid function test results in NHP using total T4 (TT4) and free T4 (FT4) assays are difficult to interpret without accurate reference intervals (RI) for comparison. As a consequence, hypothyroidism may be underdiagnosed in these species. The objective of this study was to establish RI for TT4 and FT4 in healthy populations of cynomolgus macaques ( n = 133; age range 2.6 to 24.7 y) and rhesus macaques ( n = 172; age range 0.8 to 31.0 y). Serum samples were collected across a 14-y period during routine anesthetic events in clinically healthy animals, and TT4 and FT4 concentrations were measured using commercially available immunoassays. The RI established for TT4 and FT4 were 5.1 to 14.9 ug/dL and 0.48 to 1.17 ng/dL for cynomolgus macaques, and 3.9 to 14.7 ug/dL and 0.36 to 1.12 ng/dL for rhesus macaques. Significant differences in thyroid hormone concentrations were found between Indian and Chinese origin rhesus, and between Mauritian and other origin cynomolgus. In addition, juvenile and subadult rhesus exhibited significantly higher FT4 and TT4 concentrations than did older animals. Individual RI were established for subgroups with adequately different thyroid hormone concentrations. These results will allow a more thorough diagnostic evaluation of cynomolgus and rhesus macaques with clinical signs consistent with thyroid disease and will ultimately be a refinement in NHP medicine.

The Rab32/BLOC-3-dependent pathway mediates host defense against different pathogens in human macrophages.

Macrophages provide a first line of defense against microorganisms, and while some mechanisms to kill pathogens such as the oxidative burst are well described, others are still undefined or unknown. Here, we report that the Rab32 guanosine triphosphatase and its guanine nucleotide exchange factor BLOC-3 (biogenesis of lysosome-related organelles complex-3) are central components of a trafficking pathway that controls both bacterial and fungal intracellular pathogens. This host-defense mechanism is active in both human and murine macrophages and is independent of well-known antimicrobial mechanisms such as the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent oxidative burst, production of nitric oxide, and antimicrobial peptides. To survive in human macrophages, Salmonella Typhi actively counteracts the Rab32/BLOC-3 pathway through its Salmonella pathogenicity island-1-encoded type III secretion system. These findings demonstrate that the Rab32/BLOC-3 pathway is a novel and universal host-defense pathway and protects mammalian species from various pathogens.