Defect of Interferon γ Leads to Impaired Wound Healing through Prolonged Neutrophilic Inflammatory Response and Enhanced MMP-2 Activation.

Interferon (IFN)-γ is mainly secreted by CD4+ T helper 1 (Th1), natural killer (NK) and NKT cells after skin injury. Although IFN-γ is well known regarding its inhibitory effects on collagen synthesis by fibroblasts in vitro, information is limited regarding its role in wound healing in vivo. In the present study, we analyzed how the defect of IFN-γ affects wound healing. Full-thickness wounds were created on the backs of wild type (WT) C57BL/6 and IFN-γ-deficient (KO) mice. We analyzed the percent wound closure, wound breaking strength, accumulation of leukocytes, and expression levels of COL1A1, COL3A1, and matrix metalloproteinases (MMPs). IFN-γKO mice exhibited significant attenuation in wound closure on Day 10 and wound breaking strength on Day 14 after wound creation, characteristics that are associated with prolonged neutrophil accumulation. Expression levels of COL1A1 and COL3A1 mRNA were lower in IFN-γKO than in WT mice, whereas expression levels of MMP-2 (gelatinase) mRNA were significantly greater in IFN-γKO than in WT mice. Moreover, under neutropenic conditions created with anti-Gr-1 monoclonal antibodies, wound closure in IFN-γKO mice was recovered through low MMP-2 expression levels. These results suggest that IFN-γ may be involved in the proliferation and maturation stages of wound healing through the regulation of neutrophilic inflammatory responses

Critical role of tumor necrosis factor-α in the early process of wound healing in skin

Tumor necrosis factor (TNF)-α is quickly released and initiates inflammation at wound tissues, but its precise role in wound healing is not fully understood. We examined the contribution of this cytokine to the early process of healing using a mouse model with full-thickness wounds in skin. TNF-α synthesis was detected just after wound creation, increased during the first several hours, reached a peak level on day 1, and then decreased to the basal level. In mice treated with anti-TNF-α mAb, wound closure was significantly delayed, and distances between the panniculus carnosus edges were significantly longer on day 3, compared with control. Inflammatory cell and fibroblast density were markedly decreased on day 3 in the anti-TNF-α mAb-treated mice compared with control. In contrast, wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. These results indicate that TNF-α is involved in the early process of wound healing

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

<div><p><i>Cryptococcus neoformans</i> is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against <i>Cryptococcus</i>, primarily utilizing protein-conjugated components of the <i>Cryptococcus</i> polysaccharide capsule as antigen. However, there is currently no vaccine against <i>Cryptococcus</i> in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in <i>C</i>. <i>neoformans</i> and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against <i>C</i>. <i>neoformans</i> infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, <i>Candida utilis</i>, was administered intraperitoneally, prior to infecting mice with a lethal dose of <i>C</i>. <i>neoformans</i>. GlcCer administration prevented the dissemination of <i>C</i>. <i>neoformans</i> from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of <i>C</i>. <i>neoformans</i> infection and can provide a viable vaccination strategy against <i>Cryptococcus</i>.</p></div

Detection of GlcCer antibody in the sera of different strains of mice following different route of administration.

<p>A) Sera of CBA/J mice treated with weekly administration of GlcCer + adjuvant using interaperitoneal (IP) or subcutaneous (SC) administration. *, <i>P</i> < 0.05, IgM day 28 or day 56 versus IgM normal serum (day 1). B) Sera of BALB/c mice treated with weekly administeration of GlcCer + FA. Six mice per group were used. Plots show the results of ELISA experiments performed using purified GlcCer as antigen. *, <i>P</i> < 0.05, IgM at day 14, 28, 48, or 56 versus IgM normal serum.</p

GlcCer administration results in partial immunity against cryptococcosis in mouse models of the disease.

<p>(A) Survival of CBA/J mice treated with 20 μg/day of GlcCer or GlcCer + FA and infected intranasally with 5x10⁵ <i>C</i>. <i>neoformans</i> (Cn) cells. The GlcCer and GlcCer + iFA control groups received treatment, but were not infected. The Cn group were infected, but did not receive treatment. Ten mice per group were used. *, <i>P<0</i>.<i>05</i> Cn + GlcCer or Cn + GlcCer + adjuvant versus Cn. (B) Fungal tissue burden in the lungs of the mice treated with 20 μg/day of GlcCer or GlcCer + adjuvant and infected intranasally with 5x10⁵ Cn. Three mice per group per day were used. *, <i>P<0</i>.<i>05</i>, Cn + GlcCer or Cn + GlcCer + adjuvant versus Cn H99 (C). Fungal tissue burden in the brains of the mice treated with 20 μg/day of GlcCer or GlcCer + adjuvant and infected intranasally with 5x10⁵ Cn. Three mice per group per day were used. Day = 0 represents the first day of infection. *, <i>P<0</i>.<i>05</i>, Cn + GlcCer or Cn + GlcCer = adjuvant versus Cn H99.</p

Detection of cytokines in the medium of mice liver mononuclear cells following glycolipid treatment.

<p>Detection of A) IL-4, B) IFN-ϒ. C) TNF-α, D) IL-6, E) IL-12p40, and F) IL-1β. Plots are the result of ELISA experiments performed on the media of cells after 48 hours of incubation with various concentration of β-GlcCer or 100 ng/ml of α-GalCer. * <i>P < 0</i>.<i>05</i>, GalCer versus medium, DMSO or GlcCer groups.</p

Histopathology of lungs and brain of mice infected with <i>C</i>. <i>neoformans</i> and treated with GlcCer vs. untreated mice.

<p>A) Brain of untreated mice stained with H&E. Colonization of <i>C</i>. <i>neoformans</i> (Cn) cells in the brain is shown with arrows. Histology was performed 20 days post-infection. B & C) Brain of infected mice treated with GlcCer or GlcCer + FA. Histology was performed 20 days post-infection. Images showed no abnormality in the brain tissue and no <i>C</i>. <i>neoformans</i> cells was found, confirming CFU data illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153853#pone.0153853.g002" target="_blank"><b>Fig 2C</b>, <b>2D</b> and <b>2G</b></a>) Lungs of untreated mice stained with H&E or mucicarmine. Histology was performed 20 days post-infection. Significant lung inflammation is present (arrowheads in D), where the lung tissue is infiltrated with numerous macrophages, lymphocytes and neutrophils. <i>C</i>. <i>neoformans</i> cells are readily visible in these areas (not shown) and in the alveoli (arrows in G). E & H) Lungs of mice treated with GlcCer and stained with H&E or mucicarmine. Histology was performed 90 days post-infection. Arrows in E illustrate normal lung structure. F & I) Lung of mice treated with GlcCer + adjuvant and stained with H&E or mucicarmine. Histology was performed 90 days post-infection. Arrows in F illustrate normal lung structure. Arrowheads in F illustrate some lung inflammation with no <i>C</i>. <i>neoformans</i> cells present at this particular site of inflammation (I), although they were present in other fields. Three mice per group were used in all histology experiments. The images shown are representative fields of the entire organ. Black bar in A, B an C, 200 μm; black bar in D, E and F, 200 μm; black bar in G, H and I, 40 μm.</p

Structure and composition of the analyzed GlcCer’s of <i>Candida utilis</i>.

<p>(A) Structure of GlcCer: i) GlcCer with 4,8-Sphingadienine (d18:2) sphingoid base. ii) GlcCer with 9-Methyl-4,9-Sphingadienine (d19:2) sphingoid base. The additional carbon chain, R = C9 –C27. (B) Composition analysis of GlcCer as analyzed by ESI-MS. The values are Mean ± SEM and n = 3, where ‘n’ represents analysis from 3 independent purifications. Complete list of data points in presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153853#pone.0153853.s004" target="_blank">S1 Table</a></b>.</p