Adjusted^a association (using parsimonious model) between sexual behavior and HIV sero-positivity among MSM attending HIV sentinel sites in West Bengal, 2010–11 (N = 1237).

<p>^a Adjusted for age group, current place of residence, literacy status, occupation, duration of stay in current place of residence and reason for coming to the service point</p><p>^b CL: confidence limits</p><p>* p value indicates statistically significant association with HIV sero-positivity at α = 0.05</p><p>Adjusted<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127232#t003fn001" target="_blank">^a</a> association (using parsimonious model) between sexual behavior and HIV sero-positivity among MSM attending HIV sentinel sites in West Bengal, 2010–11 (N = 1237).</p

Distribution of socio-demographic, behavioral factors and HIV sero-status among MSM attending HIV sentinel sites in West Bengal, 2010–11 (N = 1237).

<p>^a Category excludes missing values</p><p>^b Panthi: Self-identified subgroup of MSM who are externally more masculine, and usually play the insertive partner’s role during anal intercourse with another MSM¹⁴</p><p>^cDouble decker: Self-identified subgroup of MSM whose gender identity is often neutral and dependant on the identity of their partner, hence they get engaged in both (insertive as well as receptive) roles during anal intercourse with another MSM¹⁴</p><p>^dKothi: Self-identified subgroup of MSM who are more effeminate and commonly play the receptive role during anal intercourse with another MSM¹⁴</p><p>Distribution of socio-demographic, behavioral factors and HIV sero-status among MSM attending HIV sentinel sites in West Bengal, 2010–11 (N = 1237).</p

Unadjusted and adjusted^a association (using saturated model) of socio-demographic and behavioral characteristics with HIV sero-positivity among MSM attending HIV sentinel sites in West Bengal, 2010–11 (N = 1237).

<p>^a Each socio-demographic and behavioral characteristic adjusted for all others.</p><p>^b CL: confidence limits</p><p>* p value indicates statistically significant association with HIV sero-positivity at α = 0.05</p><p>Unadjusted and adjusted<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127232#t002fn001" target="_blank">^a</a> association (using saturated model) of socio-demographic and behavioral characteristics with HIV sero-positivity among MSM attending HIV sentinel sites in West Bengal, 2010–11 (N = 1237).</p

Cell-intrinsic sphingosine kinase 2 promotes macrophage polarization and renal inflammation in response to unilateral ureteral obstruction

<div><p>Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the <i>in vivo</i> significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of <i>Sphk2</i>^<i>-/-</i> mice showed reduced renal damage and diminished levels of the renal injury markers TGF<b>β</b>₁ and <b>α</b>SMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed <i>Sphk2</i>^<i>-/-</i> kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNF<b>α</b>, CXCL1 and IL<b>β</b>₁, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in <i>Sphk2</i>^<i>-/-</i> bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2^-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.</p></div

Sphk2 inhibitor treatment diminishes renal injury.

<p>6–8 week old WT mice were treated with the Sphk2 inhibitor (SK2i) at 3mg/kg. Renal injury was assessed in H&E stain (A), PAS stain (B) and scored (C). Myofibroblast infiltration indicated by protein levels of <b>α</b>SMA is significantly lower in SK2i treated mice (D), protein bands quantified using Image J and normalized to GAPDH (E). **p<0.01; *p<0.05. N = 6. Experiments repeated at least two times with N = 6 each time. Original magnification; 20x objective. Scale bar; 50μm.</p

Reduced EMT transition in absence of Sphk2 following renal injury.

<p>6–8 week old WT, Sphk2^-/- or WT mice were treated with Sphk2 inhibitor (SK2i) at 3mg/kg or vehicle control, daily by i.p three days prior to and following the UUO surgery (6 days total) and expression of EMT markers were assessed in whole kidney lysates. Western Blot analysis indicated reduced expression of EMT markers Vimentin and Fibronectin in Sphk2^-/- (A, B) and SK2i treated mice (C, D), protein bands were quantified using Image J and normalized to GAPDH. **p<0.01; *p<0.05. N = 3.</p

Diminished renal fibrosis in <i>Sphk2</i>^<i>-/-</i> and SK2i treated mice.

<p><i>Sphk2</i>^<i>-/-</i> mice and WT treated with SK2i were subjected to UUO for 7 days with appropriate control groups. Masson’s Trichrome staining indicates reduced renal fibrosis after 7 days of obstruction in <i>Sphk2</i>^<i>-/-</i> (A&B) and SK2i treated mice (C&D) compared non-obstructed controls and quantitated by Image J. N = 6. (Data represents average of 3 experiments, n = 3/experiment). **p<0.01). Original magnification; 20x objective. Scale bar; 50μm.</p

Diminished renal injury in <i>Sphk2</i>^<i>-/-</i> mice after UUO.

<p>(A) Paraffin sections were stained with Hematoxylin & Eosin (H&E) and Periodic Acid Schiff (PAS). Representative photomicrographs taken with Zeiss microscope using 40x objective, WT and <i>Sphk2</i>^<i>-/-</i> mice (N = 6) non-obstructed (NO) and obstruced (O) kidneys at day 3 and 5. H&E (A), PAS (B). Infiltration of inflammatory cells, tubular atrophy and widening of interstitial spaces (by H&E stain), and tubular damage (by PAS stain) were considered as the criteria to assess the renal injury. (C) PAS stained slides were blinded and scored for renal injury. A scale of 0–5, zero being the least and 5 being the maximum injury, revealed that <i>Sphk2</i>^<i>-/-</i> mice had diminished renal injury after 3 days of obstruction compared WT (**p<0.01). Experiments were repeated at least two times with N = 6 each time. Original magnification; 20x objective. Scale bar; 50μm.</p

Inflammatory cytokines are diminished in mice treated with SK2i.

<p>Mice subjected to UUO were treated with either vehicle (2%cyclodextrin) or SK2i (3mg/kg) and kidney tissue was harvested for qRT PCR analysis for inflammatory cytokine mRNA shows that expression levels of IL1<b>β</b>, TNF<b>α</b>, MCP-1 and CXCL1 were significantly lower in SK2i compared to vehicle treated group, N = 6. (B) Flow Cytometry analysis of isolated kidney cells show that the percentage CD11b⁺F4/80⁺ cells did not change in vehicle and SK2i treated mice whereas the proportion of anti-inflammatory M2 cells (F4/80⁺ CD11b⁺ CD206⁺) is significantly higher (C) in the SK2i group. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194053#pone.0194053.g007" target="_blank">Fig 7C</a> shows pseudo color plots for CD206⁺ cells of vehicle and SK2i treated mice and isotype control. (**p<0.01; *p<0.05). (Data represents average of 3 experiments, n = 3/experiment).</p

Sphk2 in both immune cells and kidney microenvironment contribute to renal fibrosis.

<p>(A). Recipient wild-type and <i>Sphk2</i>^-/- mice were irradiated and injected with 1 × 10⁶ bone marrow cells obtained from WT or <i>Sphk2</i>^-/- mice by tail vein. 8 weeks after engraftment mice were subjected to UUO for 7 days and trichrome staining performed on kidney sections. Image J quantification (B) indicates that WT mice receiving WT cells have the highest degree of renal fibrosis, followed by <i>Sphk2</i>^<i>-/-</i> recipients receiving WT cells and WT mice receiving <i>Sphk2</i>^<i>-/-</i> bone marrow. <i>Sphk2</i>^<i>-/-</i> mice receiving <i>Sphk2</i>^<i>-/-</i> transplanted cells had the least fibrosis. N = 4/genotype. **p<0.01; *p<0.05. Original magnification; 20x objective. Scale bar; 50μm.</p