Madhulika Dixit

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of β-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin–integrin interaction.

Objective: The underlying problem in lymphatic filariasis is irreversible swelling of the limbs (lymphoedema), which is a unique feature of lymphatic insufficiency. It is still unclear whether the natural ability of lymphatics to form functional lymphatic vasculature is achieved or attenuated in the lymphoedemal pathology. Clinical studies have clearly shown that circulating lymphatic progenitors (CLPs), a subset of bone marrow-derived mononuclear cells (PBMCs), contribute to post-natal lymph vasculogenesis. CLP-based revascularisation could be a promising strategy to bypass the endothelial disruption and damage incurred by the filarial parasites. Thus our aim was to compare and characterise the functional prowess of PBMCs in physiological and lymphoedemal pathology. Methods: PBMCs were isolated from venous blood sample from drug-naive endemic normals (EN) and drug-deprived filarial lymphoedema (FL) individuals using density gradient centrifugation. Adhesion, transwell migration and in vitro matrigel assays were employed to characterise the lymphvasculogenic potential of PBMCs. CLPs were phenotypically characterised using flow cytometry; expression levels of lymphatic markers and inflammatory cytokines were quantified using qRT-PCR and ELISA, respectively. Results: PBMCs from FL group display poor adherence to fibronectin (P = 0.040), reduced migration towards SDF-1α (P = 0.035), impaired tubular network (P = 0.004) and branching point (P = 0.048) formation. The PBMC mRNA expression of VEGFR3 (P = 0.039) and podoplanin (P = 0.050) was elevated, whereas integrin α9 (P = 0.046) was inhibited in FL individuals; additionally, the surface expression of CD34 (P = 0.048) was significantly reduced in the FL group compared to the EN group. Conclusion: PBMCs from filarial lymphoedema show defective and dysregulated lymphvasculogenic function compared to endemic normals.

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Coimmunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxiainduced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 μM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

Background and objective: Metabolic disorders including diabetes are associated with immune cell dysfunction. However, the effect of normal glucose metabolism or impairment thereof on immune cell gene expression is not well known. Hence, in this cross-sectional pilot study, we sought to determine the differences in gene expression in the peripheral blood mono-nuclear cells (PBMCs) of normal glucose tolerant (NGT) and prediabetic (PD) Asian Indian men, at fasting and in response to 75 g oral glucose load. Methods: Illumina HT12 bead chip-based microarray was performed on PBMCs at fasting and 2-h post load conditions for NGT (N = 6) and PD (N = 9) subjects. Following normalization and due quality control of the raw data, differentially expressed genes (DEGs) under different conditions within and across the two groups were identified using GeneSpring GX V12.0 software. Paired and unpaired Student's t-tests were applied along with fold change cut-offs for appropriate comparisons. Validation of the microarray data was carried out through real-time qPCR analysis. Significantly regulated biological pathways were analyzed by employing DEGs and DAVID resource. Deconvolution of the DEGs between NGT and PD subjects at fasting was performed using CIBERSORT and genes involved in regulatory T-cell (Treg) function were further analyzed for biological significance. Results: Glucose load specifically altered the expression of 112 genes in NGT and 356 genes in PD subjects. Biological significance analysis revealed transient up-regulation of innate and adaptive immune response related genes following oral glucose load in NGT individuals, which was not observed in PD subjects. Instead, in the PD group, glucose load led to an increase in the expression of pro-atherogenic and anti-angiogenic genes. Comparison of gene expression at fasting state in PD versus NGT revealed 21,707 differentially expressed genes. Biological significance analysis of the immune function related genes between these two groups (at fasting) revealed higher gene expression of members of the TLR signaling, MHC class II molecules, and T-cell receptor, chemotaxis and adhesion pathways in PD subjects. Expression of interferon-γ (IFN-γ) and TNFα was higher and that of type-1 interferons and TGF-β was lower at fasting state in PD subjects compared to NGT. Additionally, expression of multiple proteasome subunits and protein arginine methyl transferase genes (PRMTs) were higher and that of Treg specific genes was significantly distinct at fasting in PD subjects compared to NGT. Conclusion: Prediabetes uncovers constitutive TLR activation, enhanced IFN-γ signaling, and Treg dysfunction at fasting along with altered gene expression response to oral glucose load.

Aim: To compare the adhesion, migration and endothelial differentiation potential of peripheral blood-derived mononuclear cells (PBMCs) obtained from drug-naive normal glucose tolerance (NGT) and impaired glucose tolerance (IGT) Asian Indian men.

Vasoplegia observed post cardiopulmonary bypass (CPB) is associated with substantial morbidity, multiple organ failure and mortality. Circulating counts of hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPC) are potential markers of neo-vascularization and vascular repair. However, the significance of changes in the circulating levels of these progenitors in perioperative CPB, and their association with post-CPB vasoplegia, are currently unexplored. We enumerated HSC and EPC counts, via flow cytometry, at different time-points during CPB in 19 individuals who underwent elective cardiac surgery. These 19 individuals were categorized into two groups based on severity of post-operative vasoplegia, a clinically insignificant vasoplegic Group 1 (G1) and a clinically significant vasoplegic Group 2 (G2). Differential changes in progenitor cell counts during different stages of surgery were compared across these two groups. Machine-learning classifiers (logistic regression and gradient boosting) were employed to determine if differential changes in progenitor counts could aid the classification of individuals into these groups. Enumerating progenitor cells revealed an early and significant increase in the circulating counts of CD34+ and CD34+CD133+ hematopoietic stem cells (HSC) in G1 individuals, while these counts were attenuated in G2 individuals. Additionally, EPCs (CD34+VEGFR2+) were lower in G2 individuals compared to G1. Gradient boosting outperformed logistic regression in assessing the vasoplegia grouping based on the fold change in circulating CD 34+ levels. Our findings indicate that a lack of early response of CD34+ cells and CD34+CD133+ HSCs might serve as an early marker for development of clinically significant vasoplegia after CPB.