A systemic inflammatory response (SIR) strongly modulates CHI3L1, CHIA and CHIT expression in a mouse model of burn injury and sepsis.

<p>WT mice were exposed to burn injury (20% <u>B</u>ody <u>S</u>urface <u>A</u>rea), lipopolysaccharide (LPS) or oral gavage with Pseudomonas strain (PA14) 48 hours prior to tissue analysis. (A) CHI3L1 expression was strongly induced in lung tissue under all conditions of SIR but also in colon following exposure to PA14 strain. (B) CHIA was predominately induced in liver but also colon following exposure to PA14 strain. (C) CHIT1 appeared to be induced in spleen and liver by burn injury, with or without LPS and PA14 exposure (all: qPCR /ΔΔCt-method: n≥4; mean±SD; normalized to respective organ control).</p

Induced enteric chitinase expression likely promotes entero-hematogenic bacterial translocation following burn injury in a mouse model of enteric colonization with PA14 strain.

<p>Experimental design: (Step 1) Acidified drinking water was supplemented with chitosan (50mg/l) four weeks prior to experiments while controls continued to receive unsupplemented acidified water. (Step 2) 48 hours prior to burn injury, enteric colonization with PA14 strain of WT mice was performed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140440#pone.0140440.s003" target="_blank">S3 Fig</a>). (Step 3) With burn injury (full thickness, dorsal skin fold, ~20% BSA), (A) the Kaplan–Meier estimator of survival suggested a higher incidence of lethal PA14 septicemia in animals pre-sensitized with chitosan in drinking water (Burn+PA14 v.s. Burn+PA14+ chitosan-pre-sensitized: n = 12, χ² = 0.39, P = 0.53, hazard ratio = 0.84). (B) Cell count of peritoneal lavage (mostly neutrophils) suggested burn-induced peritonitis due to enteric translocation of PA14 strain, with or without enteric chitinase induction. (C) Similarly, blood culture suggested burn-induced entero-hematogenic translocation of PA14 strain. (B & C: n≥10; mean±SD; *P<0.05; normalized to control)</p

<b>Schematic of the effects of Foxn1 loss-of-function on keratinocytic lineage differentiation in nude mice.</b>

<p>(A) A severe disturbance in keratin synthesis and terminal differentiation in the Foxn1^−/− phenotype also affects upstream lineage progenitors with strong upregulation of Lhx2 as a key feature. (B) Altered Wnt- and Shh-signaling in the Foxn1^−/− phenotype.</p

Exposure to chitosan induces chitinase expression in skin and colon.

<p>WT mice were exposed to preparations of chitosan (poly-D-glucosamine) s.c. (3x250μg/100μl over 2 weeks) and via drinking water (50mg/l over 2 weeks). (A) With s.c. injection, only CHIA was significantly induced in skin. (B) Chitosan in drinking water induced all chitinases, mostly CHI3L1 (all: qPCR /ΔΔCt-method: n≥4; mean±SD; *P<0.05; normalized to respective organ control).</p

A modulation of chitinase expression in skin increases survival rates in a mouse model of opportunistic wound infection with PA14 strain.

<p>Experimental design: (Step 1) WT mice were injected low-dose s.c. with PA14 strain (10¹ CFU/100μl; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140440#pone.0140440.s002" target="_blank">S2 Fig</a>) four weeks prior to experiments in order to induce a uniform PA14-specific immunity. (Step 2) For an additional two weeks animals received s.c.-injections of chitosan (3x250μg/100μl) while the control groups received vehicle in order to induce the expression of chitinases in skin. (Step 3) At 48h post-burn injury (full thickness, dorsal skin fold, ~20% BSA), the burn area was debrided and contaminated with 10⁴ CFU/10μl PA14 strain. We saw that animals pre-sensitized with chitosan were less likely to develop lethal PA14 septicemia (PA14 control v.s. PA14 chitosan-pre-sensitized: n = 10, χ² = 0.31, P = 0.58, hazard ratio = 1.74).</p

CHI3L1, CHIA and CHIT show distinct, organ-specific expression levels in wild type (WT) mice.

<p>(A) Intra-organ comparison regarding skin, spleen, lungs, liver or colon demonstrates pre-dominance of CHI3L1 expression levels over CHIA and CHIT. (B) Inter-organ comparison of CHI3L1, CHIA or CHIT each also reveals an apparent biological relevance e.g. of CHI3L1-expression in lung tissue (qPCR /ΔΔCt-method: n≥4; normalized to lowest expression value of each data set = fold change).</p

<b>Schematic of mature, anagen hair follicle (HF) morphology with typical localization of keratinocytic lineage progeny in wild type (WT).</b>

<p>CD49f/α6-integrin (blue) positive epithelium dominates within the basal layer of the surface epithelium. CD200 (purple) and CD34 (red) are both strongly expressed within the bulge region. In addition, CD200 is also expressed in upper parts of the outer root sheet (ORS) whereas CD34 expression may extend below the bulge region. Matrix cells of the hair bulb are considered CD49f/CD200/CD34 negative. In general, surface markers of epithelial progeny are not exclusive for each other in part depending on the stage of hair cycling. This study evaluates various effects of Foxn1 loss-of-function on telogen-anagen hair cycling and epithelial stem cell niche regulation in Nu/Nu mice.</p

<b>Comparative expression of stem cell markers in the Nu/Nu phenotype vs. WT.</b>

<p>(A) Following induction of a new anagen hair cycle by depilation, WT skin shows a distinct dynamic expression of the embryonic stem cell (SC) markers Sox2 and Oct4. (B) Conversely, in the Nu/Nu phenotype, both markers are permanently suppressed (qPCR/ΔΔCt-method: n = 4; *P<0.05). (C) Other epithelial lineage SC markers show distinct changes in expression compared to WT, notably of Lhx2. (D) IHC confirms a very localized Lhx2 expression in the bulge region (asterisk) of WT telogen (D1) HF’s that extents to the infundibulum and the epithelial surface (arrow) during anagen (D2/3). In the Nu/Nu phenotype Lhx2 is strongly expressed in both hyperplastic surface epithelium and bulge region proximity. Scale bars: 100 µm (D).</p

<b>Morphological features along with CyclinD1 levels suggest a continuous, yet frustrated activation of hair cycling within the nude mouse phenotype.</b>

<p>(A) The Foxn1^−/− (Nu/Nu) phenotype demonstrates spontaneous lanugo hair growth as opposed to a complete hairless condition. Histologically, superficial epithelial cysts/pseudo-infundibula which often fail to connect to the surface and associate with sebaceous gland cells (1) represent the equivalent of HF’s in wild type (WT). In addition, invaginations of a hyperplastic epithelium (2) resemble stages of de novo embryonic HF formation. With spontaneous lanugo hair formation, a dysmorphic root sheet (RS) (3) and infundibulum (4) containing a rudimentary hair shaft can be seen. (B) In WT telogen, a dermal papilla complex (5) gets activated by depilation and a newly forming RS expands both in width and length into the deeper dermis (6) in order to generate several terminal hair shafts. (C) Comparative CyclinD1 mRNA levels are suggestive for a telogen and anagen equivalent in the Nu/Nu phenotype (qPCR/ΔΔCt-method: n = 6; *P<0.05). The red horizontal line represents the telogen skin level used as reference. Scale bars: 4 mm (macroscopic); 50 µm (microscopic).</p

<b>As Oct3/4+ population is expanded; CD49f+, CD34+ and CD200+ populations are reduced in epithelial isolates from newborn skin of the Foxn1^−/− phenotype vs. wild type.</b>

<p>(A) Flow cytometry summary table listing fractions positive for markers of progeny within epithelial isolates from newborns (%±SD, n>6). Distinct differences between the Foxn1^−/− phenotype and wild type are demonstrated by comparative sample flow cytometry data gated for (B) CD49f, CD34, CD200 and (C) Oct3/4 positive epithelial fractions.</p