Analysis of the genes controlling cotton fiber length reveals the molecular basis of plant breeding and the genetic potential of current cultivars for continued improvement.

Stagnated crop improvement has raised questions of whether and how current crop cultivars can be further improved. Genes are the core determinants of performance of all cultivars. Here, we report the molecular basis of plant breeding and address these questions by analyzing 226 GFL genes controlling and accurately predicting fiber length, an important breeding objective trait, in cotton (Gossypium sp.). We first identified the favorable allele and the number of favorable alleles (NFAs) of each GFL gene, calculated the total NFAs of the 226 GFL genes accumulated in 198 advanced breeding lines, and analyzed them against fiber lengths. Fiber lengths of the breeding lines were strongly correlated with the total NFAs of the GFL genes (r = 0.85, P < 0.0001), suggesting that accumulation of the favorable alleles of the genes controlling objective traits is the molecular basis of cotton breeding. Surprisingly, a breeding line with a fiber length of present cultivars having the longest fibers contained only about 51% of the total NFAs of the 226 GFL genes. The genetic potentials of current cultivars were then predicted using linear and non-linear models, respectively, revealing that a breeding line or cultivar with a fiber length of 33.8 mm could be further improved in fiber length by up to 118%. Finally, we showed that the genetic potential of such a breeding line can be realized through gene-based breeding. Therefore, these findings shed light on continued crop improvement in general and provide 740 genic biomarkers desirable for enhanced cotton fiber breeding.

Accurate prediction of complex traits for individuals and offspring from parents using a simple, rapid, and efficient method for gene-based breeding in cotton and maize.

Accurate, simple, rapid, and inexpensive prediction of complex traits controlled by numerous genes is paramount to enhanced plant breeding, animal breeding, and human medicine. Here we report a novel method that enables accurate, simple, and rapid prediction of complex traits of individuals or offspring from parents based on the number of favorable alleles (NFAs) of the genes controlling the objective traits. The NFAs of 226 cotton fiber length (GFL) genes and nine maize hybrid grain yield related (ZmF1GY) genes were directly used to predict cotton fiber lengths of individual plants and maize grain yields of F1 hybrids from parents, respectively, using prediction model-based methods as controls. The NFAs of the 226 GFL genes predicted cotton fiber lengths at an accuracy of 0.85, as the model methods and outperforming genomic prediction by 82 % - 170 %. The NFAs of the nine ZmF1GY genes predicted grain yields of maize hybrids from parents at an accuracy of 0.80, outperforming genomic prediction by 67 %. Moreover, the prediction accuracies of these traits were consistent across years, environments, and eco-agricultural systems. Importantly, the accurate prediction of these traits directly using the NFAs of the genes allows breeding to be performed in greenhouse, phytotron, or off-season, without the need of the model training and validation steps essential and costly for model-based genomic or genic prediction. Therefore, this new method dramatically outperforms the current model-based genomic methods used for phenotype prediction and streamlines the process of breeding, thus promising to substantially enhance current plant and animal breeding.

Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils, and Mast Cell Degranulation.

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

An Epithelial Abrasion Model for Studying Corneal Wound Healing.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Accurate Prediction of a Quantitative Trait Using the Genes Controlling the Trait for Gene-Based Breeding in Cotton.

Accurate phenotype prediction of quantitative traits is paramount to enhanced plant research and breeding. Here, we report the accurate prediction of cotton fiber length, a typical quantitative trait, using 474 cotton (Gossypium ssp.) fiber length (GFL) genes and nine prediction models. When the SNPs/InDels contained in 226 of the GFL genes or the expressions of all 474 GFL genes was used for fiber length prediction, a prediction accuracy of r = 0.83 was obtained, approaching the maximally possible prediction accuracy of a quantitative trait. This has improved by 116%, the prediction accuracies of the fiber length thus far achieved for genomic selection using genome-wide random DNA markers. Moreover, analysis of the GFL genes identified 125 of the GFL genes that are key to accurate prediction of fiber length, with which a prediction accuracy similar to that of all 474 GFL genes was obtained. The fiber lengths of the plants predicted with expressions of the 125 key GFL genes were significantly correlated with those predicted with the SNPs/InDels of the above 226 SNP/InDel-containing GFL genes (r = 0.892, P = 0.000). The prediction accuracies of fiber length using both genic datasets were highly consistent across environments or generations. Finally, we found that a training population consisting of 100-120 plants was sufficient to train a model for accurate prediction of a quantitative trait using the genes controlling the trait. Therefore, the genes controlling a quantitative trait are capable of accurately predicting its phenotype, thereby dramatically improving the ability, accuracy, and efficiency of phenotype prediction and promoting gene-based breeding in cotton and other species.

Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

Corneal dysfunction precedes the onset of hyperglycemia in a mouse model of diet-induced obesity.

PURPOSE: The purpose of this study was to use a mouse model of diet-induced obesity to determine if corneal dysfunction begins prior to the onset of sustained hyperglycemia and if the dysfunction is ameliorated by diet reversal. METHODS: Six-week-old male C57BL/6 mice were fed a high fat diet (HFD) or a normal diet (ND) for 5-15 weeks. Diet reversal (DiR) mice were fed a HFD for 5 weeks, followed by a ND for 5 or 10 weeks. Corneal sensitivity was determined using aesthesiometry. Corneal cytokine expression was analyzed using a 32-plex Luminex assay. Excised corneas were prepared for immunofluorescence microscopy to evaluate diet-induced changes and wound healing. For wounding studies, mice were fed a HFD or a ND for 10 days prior to receiving a central 2mm corneal abrasion. RESULTS: After 10 days of HFD consumption, corneal sensitivity declined. By 10 weeks, expression of corneal inflammatory mediators increased and nerve density declined. While diet reversal restored nerve density and sensitivity, the corneas remained in a heightened inflammatory state. After 10 days on the HFD, corneal circadian rhythms (limbal neutrophil accumulation, epithelial cell division and Rev-erbα expression) were blunted. Similarly, leukocyte recruitment after wounding was dysregulated and accompanied by delays in wound closure and nerve recovery. CONCLUSION: In the mouse, obesogenic diet consumption results in corneal dysfunction that precedes the onset of sustained hyperglycemia. Diet reversal only partially ameliorated this dysfunction, suggesting a HFD diet may have a lasting negative impact on corneal health that is resistant to dietary therapeutic intervention.

Analysis of the genes controlling three quantitative traits in three diverse plant species reveals the molecular basis of quantitative traits.

Most traits of agricultural importance are quantitative traits controlled by numerous genes. However, it remains unclear about the molecular mechanisms underpinning quantitative traits. Here, we report the molecular characteristics of the genes controlling three quantitative traits randomly selected from three diverse plant species, including ginsenoside biosynthesis in ginseng (Panax ginseng C.A. Meyer), fiber length in cotton (Gossypium hirsutum L. and G. barbadense L.) and grain yield in maize (Zea mays L.). We found that a vast majority of the genes controlling a quantitative trait were significantly more likely spliced into multiple transcripts while they expressed. Nevertheless, only one to four, but not all, of the transcripts spliced from each of the genes were significantly correlated with the phenotype of the trait. The genes controlling a quantitative trait were multiple times more likely to form a co-expression network than other genes expressed in an organ. The network varied substantially among genotypes of a species and was associated with their phenotypes. These findings indicate that the genes controlling a quantitative trait are more likely pleiotropic and functionally correlated, thus providing new insights into the molecular basis underpinning quantitative traits and knowledge necessary to develop technologies for efficient manipulation of quantitative traits.

Serial block-face scanning electron microscopy reveals neuronal-epithelial cell fusion in the mouse cornea.

The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8-10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ≤ 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation.

Dyslipidemia and Meibomian Gland Dysfunction: Utility of Lipidomics and Experimental Prospects with a Diet-Induced Obesity Mouse Model.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease and loss of ocular surface homeostasis. Increasingly, several observational clinical studies suggest that dyslipidemia (elevated blood cholesterol, triglyceride or lipoprotein levels) can initiate the development of MGD. However, conclusive evidence is lacking, and an experimental approach using a suitable model is necessary to interrogate the relationship between dyslipidemia and MGD. This systematic review discusses current knowledge on the associations between dyslipidemia and MGD. We briefly introduce a diet-induced obesity model where mice develop dyslipidemia, which can serve as a potential tool for investigating the effects of dyslipidemia on the meibomian gland. Finally, the utility of lipidomics to examine the link between dyslipidemia and MGD is considered.

Quantification of gene expression while taking into account RNA alternative splicing.

Gene expression has been widely used in functional genomics research; however, the gene expressions quantified with different methods have been frequently inconsistent, thus challenging the conclusions from such research. Here we have addressed this issue, while taking into account RNA alternative splicing. We found that when a gene was subjected to RNA alternative splicing, it was impossible or difficult to properly quantify the expression of a transcript of the gene or its overall expression using quantitative real-time PCR (qPCR), Northern hybridization, microarray, or serial analysis of gene expression. Shot-gun RNA-seq was the most proper to quantify the expression of a transcript or a gene in such cases. Moreover, the expressions of individual transcripts quantified by shot-gun RNA-seq were highly reproducible (r = 0.90-0.98) between individuals. Therefore, shot-gun or full-length RNA-seq should be the method of choice to properly quantify the expression of a transcript or a gene.

Regulation of the ovarian inflammatory response at ovulation by nuclear progesterone receptor.

PROBLEM: The nuclear progesterone receptor (PGR) transcription factor is essential for ovulation; however, the exact mechanisms by which PGR controls ovulation are not known. The aim of this study was to determine whether PGR regulates inflammatory mediators in the ovary. METHOD OF STUDY: Ovaries from mice lacking PGR (PRKO) and heterozygous PR+/- littermates were subjected to microarray analysis of a large panel of inflammatory genes. Immune cell subsets were detected by gene expression; and neutrophils by immunohistochemistry and chemotaxis assay. RESULTS: PRKO ovaries exhibited dysregulated expression of vasodilator (Edn1), cytokine (Il-6, Tgfb1), adhesion receptor (Cd34), apoptotic factor (Bax) and transcription factors (Nfkb2, Socs1, Stat3). Ptgs2 was also reduced in PRKO ovaries, but mRNA and protein were not different in granulosa cells. There were reduced neutrophils in ovaries of PRKO mice at ovulation; however, chemotaxis assays showed PRKO neutrophils migrate normally and that PRKO ovarian extracts exhibit chemotactic properties in vitro. CONCLUSION: Specific inflammatory mediators are altered in the ovaries of PRKO mice indicating that progesterone regulates features of inflammation at ovulation.

IL-20 promotes epithelial healing of the injured mouse cornea.

After corneal epithelial injury, the ensuing inflammatory response is necessary for efficient wound healing. While beneficial healing effects are attributed to recruited neutrophils and platelets, dysregulated inflammation (too little or too much) is associated with impaired wound healing. The purpose of this study was to use an established C57BL/6J mouse model of corneal injury to evaluate the potential modulatory role of interleukin-20 (IL-20) on the inflammatory and healing responses to epithelial wounding. In the uninjured cornea, immunofluorescence staining for IL-20 and its receptor, IL-20RA, was observed on basal epithelial cells at the limbus. After a 2 mm central epithelial abrasion, IL-20 staining was also observed in stromal keratocytes and ELISA studies showed a significant increase (nearly 3-fold) in IL-20 expression. Injured corneas healed more slowly when treated with a topical application of a neutralizing anti-IL-20 antibody. While corneal epithelial cell division and epithelial nerve recovery measured at 24 h post-injury were reduced compared to controls, neutrophil influx into the cornea was increased. In contrast, topical application of recombinant IL-20 (rIL-20) decreased corneal inflammation as evidenced by reductions in limbal vessel dilatation, platelet extravasation, neutrophil recruitment and CXCL1 expression. In wild type mice, topical rIL-20 had a limited effect on corneal wound healing and resulted in only a slight increase in epithelial cell division and epithelial nerve recovery; the rate of wound closure was unaffected. To clarify the effect of IL-20 on corneal wound healing, rIL-20 was topically applied to neutropenic wild type (WT) mice and mutant mice (ɣδ T cell deficient mice and CD11a deficient mice), all of which have well characterized reductions in neutrophil recruitment and delayed wound healing after corneal injury. In each case, rIL-20 restored corneal wound healing to baseline levels while neutrophil recruitment remained low. Thus, it appears that IL-20 plays a beneficial and direct role in corneal wound healing while negatively regulating neutrophil and platelet infiltration.

Ghrelin receptor regulates adipose tissue inflammation in aging.

Aging is commonly associated with low-grade adipose inflammation, which is closely linked to insulin resistance. Ghrelin is the only circulating orexigenic hormone which is known to increase obesity and insulin resistance. We previously reported that the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHS-R), increases in adipose tissues during aging, and old Ghsr(-/-) mice exhibit a lean and insulin-sensitive phenotype. Macrophages are major mediators of adipose tissue inflammation, which consist of pro-inflammatory M1 and anti-inflammatory M2 subtypes. Here, we show that in aged mice, GHS-R ablation promotes macrophage phenotypical shift toward anti-inflammatory M2. Old Ghsrp(-/-) mice have reduced macrophage infiltration, M1/M2 ratio, and pro-inflammatory cytokine expression in white and brown adipose tissues. We also found that peritoneal macrophages of old Ghsrp(-/-) mice produce higher norepinephrine, which is in line with increased alternatively-activated M2 macrophages. Our data further reveal that GHS-R has cell-autonomous effects in macrophages, and GHS-R antagonist suppresses lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Collectively, our studies demonstrate that ghrelin signaling has an important role in macrophage polarization and adipose tissue inflammation during aging. GHS-R antagonists may serve as a novel and effective therapeutic option for age-associated adipose tissue inflammation and insulin resistance.

Evaluating Pilose, a Cultigen of Gossypium hirsutum, as a Source of Resistance to Cotton Fleahopper (Hemiptera: Miridae).

Cotton fleahopper (Pseudatomoscelis seriatus Reuter) (Hemiptera: Miridae) is a piercing-sucking insect that has emerged as a major pest of cotton (Gossypium hirsutum L.) in Texas. Cotton fleahoppers feed on floral buds, commonly referred to as squares, causing damage and abscission, and subsequent yield loss. Previous studies indicate that plant resistance to cotton fleahopper is present in upland cotton, but the mechanism of resistance remains undetermined. In this study, Pilose, a cultigen of G. hirsutum, was examined as a source of resistance to cotton fleahopper, focusing on mechanism of resistance and heritability of the resistance trait. Results indicated that the resistance trait in Pilose is heritable and that pubescence is causative of resistance or that the resistance trait may be tightly linked to genes controlling pubescence. Behavioral assays indicated nonpreference as a mode of resistance in plants with dense pubescence.

Proinsulin-producing, hyperglycemia-induced adipose tissue macrophages underlie insulin resistance in high fat-fed diabetic mice.

Adipose tissue macrophages (ATMs) play an important role in the pathogenesis of obese type 2 diabetes. High-fat diet (HFD)-induced obesity has been shown to lead to ATM accumulation in rodents; however, the impact of hyperglycemia on ATM dynamics in HFD-fed type 2 diabetic models has not been studied. We previously showed that hyperglycemia induces the appearance of proinsulin (PI)-producing proinflammatory bone marrow (BM)-derived cells (PI-BMDCs) in rodents. We fed a 60% HFD to C57BL6/J mice to produce an obese type 2 diabetes model. Absent in chow-fed animals, PI-BMDCs account for 60% of the ATMs in the type 2 diabetic mice. The PI-ATM subset expresses TNF-α and other inflammatory markers, and is highly enriched within crownlike structures (CLSs). We found that amelioration of hyperglycemia by different hypoglycemic agents forestalled PI-producing ATM accumulation and adipose inflammation in these animals. We developed a diphtheria toxin receptor-based strategy to selectively ablate PI-BMDCs among ATMs. Application of the maneuver in HFD-fed type 2 diabetic mice was found to lead to near total disappearance of complex CLSs and reversal of insulin resistance and hepatosteatosis in these animals. In sum, we have identified a novel ATM subset in type 2 diabetic rodents that underlies systemic insulin resistance.

Loss of n-6 fatty acid induced pediatric obesity protects against acute murine colitis.

Dietary influences may affect microbiome composition and host immune responses, thereby modulating propensity toward inflammatory bowel diseases (IBDs): Crohn disease (CD) and ulcerative colitis (UC). Dietary n-6 fatty acids have been associated with UC in prospective studies. However, the critical developmental period when (n-6) consumption may induce UC is not known. We examined the effects of transiently increased n-6 consumption during pediatric development on subsequent dextran-sulfate-sodium (DSS)-induced acute murine colitis. The animals transiently became obese then rapidly lost this phenotype. Interestingly, mice were protected against DSS colitis 40 days after n-6 consumption. The transient high n-6-induced protection against colitis was fat type- and dietary reversal-dependent and could be transferred to germ-free mice by fecal microbiota transplantation. We also detected decreased numbers of chemokine receptor (Cxcr)5(+) CD4(+) T cells in the mesenteric lymph nodes (MLNs) of transiently n-6-fed mice. Further experiments revealed that anti-chemokine ligand (Cxcl)13 (the ligand of Cxcr5) antibody treatment decreased DSS colitis severity, implicating the importance of the Cxcr5-Cxcl13 pathway in mammalian colitis. Consecutively, we found elevated CXCL13 concentrations (CD: 1.8-fold, P = 0.0077; UC: 1.9-fold, P = 0.056) in the serum of untreated pediatric IBD patients. The human serologic observations supported the translational relevance of our findings.

Platelet recruitment promotes keratocyte repopulation following corneal epithelial abrasion in the mouse.

Corneal abrasion not only damages the epithelium but also induces stromal keratocyte death at the site of injury. While a coordinated cascade of inflammatory cell recruitment facilitates epithelial restoration, it is unclear if this cascade is necessary for keratocyte recovery. Since platelet and neutrophil (PMN) recruitment after corneal abrasion is beneficial to epithelial wound healing, we wanted to determine if these cells play a role in regulating keratocyte repopulation after epithelial abrasion. A 2 mm diameter central epithelial region was removed from the corneas of C57BL/6 wildtype (WT), P-selectin deficient (P-sel-/-), and CD18 hypomorphic (CD18hypo) mice using the Algerbrush II. Corneas were studied at 6h intervals out to 48h post-injury to evaluate platelet and PMN cell numbers; additional corneas were studied at 1, 4, 14, and 28 days post injury to evaluate keratocyte numbers. In WT mice, epithelial abrasion induced a loss of anterior central keratocytes and keratocyte recovery was rapid and incomplete, reaching ~70% of uninjured baseline values by 4 days post-injury but no further improvement at 28 days post-injury. Consistent with a beneficial role for platelets and PMNs in wound healing, keratocyte recovery was significantly depressed at 4 days post-injury (~30% of uninjured baseline) in P-sel-/- mice, which are known to have impaired platelet and PMN recruitment after corneal abrasion. Passive transfer of platelets from WT, but not P-sel-/-, into P-sel-/- mice prior to injury restored anterior central keratocyte numbers at 4 days post-injury to P-sel-/- uninjured baseline levels. While PMN infiltration in injured CD18hypo mice was similar to injured WT mice, platelet recruitment was markedly decreased and anterior central keratocyte recovery was significantly reduced (~50% of baseline) at 4-28 days post-injury. Collectively, the data suggest platelets and platelet P-selectin are critical for efficient keratocyte recovery after corneal epithelial abrasion.

γδ T cells promote inflammation and insulin resistance during high fat diet-induced obesity in mice.

γδ T cells are resident in AT and increase during diet-induced obesity. Their possible contribution to the inflammatory response that accompanies diet-induced obesity was investigated in mice after a 5 to 10 week milk HFD. The HFD resulted in significant increases in CD44(hi), CD62L(lo), and TNF-α(+) γδ T cells in eAT of WT mice. Mice deficient in all γδ T cells (TCRδ(-/-)) or only Vγ4 and Vγ6 subsets (Vγ4/6(-/-)) were compared with WT mice with regard to proinflammatory cytokine production and macrophage accumulation in eAT. Obesity among these mouse strains did not differ, but obese TCRδ(-/-) and Vγ4/6(-/-) mice had significantly reduced eAT expression of F4/80, a macrophage marker, and inflammatory mediators CCL2 and IL-6 compared with WT mice. Obese TCRδ(-/-) mice had significantly reduced CD11c(+) and TNF-α(+) macrophage accumulation in eAT after 5 and 10 weeks on the HFD, and obese Vγ4/6(-/-) mice had significantly increased CD206(+) macrophages in eAT after 5 weeks on the diet and significantly reduced macrophages after 10 weeks. Obese TCRδ(-/-) mice had significant reductions in systemic insulin resistance and inflammation in liver and skeletal muscle after longer-term HFD feeding (10 and 24 weeks). In vitro studies revealed that isolated γδ T cells directly stimulated RAW264.7 macrophage TNF-α expression but did not stimulate inflammatory mediator expression in 3T3-L1 adipocytes. These findings are consistent with a role for γδ T cells in the proinflammatory response that accompanies diet-induced obesity.

Integrin-dependent neutrophil migration in the injured mouse cornea.

As an early responder to an inflammatory stimulus, neutrophils (PMNs) must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, PMNs recruited from the peripheral limbal vasculature migrate into the avascular corneal stroma. In vitro studies suggest PMN locomotion over 2-D surfaces is dependent on integrin binding while migration within 3-D matrices can be integrin-independent. Electron micrographs of injured mouse corneas show migrating PMNs make extensive surface contact not only with collagen fibrils in the extracellular matrix (ECM), but also keratocytes. Evidence supporting involvement of integrins in corneal inflammation has prompted research and development of integrin blocking agents for use as anti-inflammatory therapies. However, the role of integrin binding (cell-cell; cell-ECM) during stromal migration in the inflamed cornea has previously not been clearly defined. In this study in vivo time lapse imaging sequences provided the means to quantify cell motility while observing PMN interactions with keratocytes and other stromal components in the living eye. The relative contribution of ß1, ß2 and ß3 integrins to PMN locomotion in the inflamed mouse cornea was investigated using blocking antibodies against the respective integrins. Of the 3 integrin families (ß1, ß2 and ß3) investigated for their potential role in PMN migration, only ß1 antibody blockade produced a significant, but partial, reduction in PMN motility. The preferential migration of PMNs along the keratocyte network was not affected by integrin blockade. Hence, the dominant mechanism for PMN motility within the corneal stroma appears to be integrin-independent as does the restriction of PMN migration paths to the keratocyte network.