Analysis of skin-barrier abnormalities.

<p><b>(A)</b> Newborn (P0) pups were analysed by barrier penetration assay using toluidine blue (TB). Remaining barrier-defect in the area of nostrils of Klk5^-/-Klk7^-/-Sp5^A135X/A135X pups in the vicinity of nostrils is marked by black arrowhead <b>(B)</b> TEWL analysis of P0 pups as a reduction of body-weight over time, n≥4 for each genotype. Error bars represent standard errors of mean; Klk/Spink5 mutant lines were compared with the wt line using Mann-Whitney U-test at 3h and 4h, ns means “not significant“; * p < 0.05 <b>(C)</b> Epidermal barrier in P5 Klk5^-/-Sp5^A135X/A135X pups was compromised in the proximity of hair follicles <b>(D)</b> Vibrissae hair (upper panel) and dorsal skin (lower panel) obtained from P5 pups analysed using scanning electron microscopy. Defective separation of hair shafts from the root sheath in Klk5^-/-Sp5^A135X/A135X is marked by white arrowhead. Dorsal skin of Klk5^-/-Sp5^A135X/A135X mice showed complete absence of hair shafts and exposed upper parts of hair follicles (yellow arrowheads); Scale bar, 300 μm.</p

Histological analysis of epidermis structure at E18.5 dpc and P0.

<p><b>(A)</b> Hematoxylin and eosin stained skin sections from newborn mice showed a reduced granular layer and acanthosis in epidermis of Sp5^A135X/A135X pups; no obvious defects were observed in the other groups, Scale bar, 100 μm. <b>(B)</b> Analysis of epidermal differentiation in the skin from E18.5 dpc embryos. Sections were stained with antibodies against keratin14 (Krt14), keratin6 (Krt6) and fillagrin (Flg). Increased expression of Krt14 was observed in Sp5^A135X/A135X embryos, which also strongly express the stress marker Krt6. Expression of both Krt6 and Krt14 was not altered in Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X. Flg staining revealed absence of profilaggrin granules in Sp5^A135X/A135X and Klk7^-/-Sp5^A135X/A135X embryos whereas these granules were present in wt, Klk5^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice (white arrowheads). Scale bar, 50 μm.</p

Generation of Spink5/Klk5/Klk7 mutant lines.

<p><b>(A)</b> Klk5^-/- line (depicted as Klk5^-/-Klk7^+/+Spink5^+/+), was used for preparation of Klk5^-/-Klk7^-/- mice (depicted as Klk5^-/-Klk7^-/-Spink5^+/+) by TALEN mutagenesis. Obtained Klk5^-/-Klk7^-/- mice were further crossed to a Flippase (FLPe) expressing mouse line to allow conditionally expressed Klk5, thus generating Klk7^-/- mice (depicted as Klk5^+/+(loxP)Klk7^-/-Spink5^+/+). Klk5^-/-, Klk7^-/- and Klk5^-/-Klk7^-/- lines were subsequently crossed with Spink5^+/- line to obtain Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X, i.e. double and triple KO lines. <b>(B)</b> Expression of Klk5, Klk7 and Spink5 at the mRNA level was quantified using qRT-PCR, n≥4 for each genotype, error bars represent standard deviations from mean.</p

MMP19^−/− mice exhibit reduced T cell activation in CHS.

<p>Using flow cytometry cells of inguinal (A and B) or draining lymph nodes (C and D) from wild-type (+/+) and MMP19-deficient (−/−) mice were analyzed for the indicated activation markers, all gated on CD8⁺ T cells. (A) Unsensitized MMP19^−/− mice show slight decrease of T cells positive for CD62L and CD95/CD25. (B) MMP19^−/− mice analyzed 24 h after abdominal FITC painting (sensitization) exhibit higher numbers of naive T cells (CD62L⁺) and decreased numbers of activated T cells compared to MMP19^+/+ mice. (C) In CHS reaction 48 h after FITC challenge on ears, draining lymph nodes of MMP19^−/− mice show significantly reduced numbers of cells positive for activation and memory markers. (D) Reduced numbers of CD25⁺ and high numbers of CD62L⁺ cells are still present in MMP19^−/− mice after 72 h while other activation markers were comparable to those of MMP19^+/+ mice. In A and B two scales are used to match relevant data from an identical experiment. Significant values (p<0.05) are marked by asterisk. Each analysis was carried out four times with MMP19^+/+ (n = 4) and MMP19^−/− mice (n = 4) per experiment.</p

Thymocytes of MMP19^−/− mice exhibit strongly reduced proliferation.

<p>(A) Immature MMP19^−/− mice exhibit low numbers of Ki-67⁺ medullar cells in the thymus. Scale bars: 50 µm. (B and C). To quantify proliferating thymocytes mice were injected i.p. with BrdU. After 14 h, CD3⁺, CD4⁺, and CD8⁺ thymocytes were analyzed for BrdU incorporation using flow cytometry. Grey, isotype control; light red, MMP19^−/− (n = 4); red, MMP19^+/+ (n = 4). (C) Quantification of BrdU positive thymocytes. CD3⁺, CD4⁺, and CD8⁺ thymocytes from MMP19^−/− mice exhibit markedly diminished proliferation. Black bars: MMP19^+/+, white bars: MMP19^−/−. (D) Quantitative RT-PCR analysis of MMP19 in the thymus shows expression in WT mice and confirmed the absence in MMP19^−/− mice. The expression of MMP19 in the thymus is lower compared to that in the liver. C_T, cycle of threshold. Significances with **p<0,01 (student's t-test).</p

Gross phenotype of Spink5- Klk- deficient mutant lines.

<p><b>(A)</b> Phenotype of wt, Sp5^A135X/A135X, Klk5^-/-Sp5^A135X/A135X, Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice 12 hours after birth. Peeling skin was observed in Sp5^A135X/A135X and Klk7^-/-Sp5^A135X/A135X and to lesser extent in Klk5^-/-Sp5^A135X/A135X pups (black arrowheads). <b>(B)</b> wt, Klk5^-/-Sp5^A135X/A135X, and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at P5. Klk5^-/-Sp5^A135X/A135X pups showed dry, scaly skin while Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice had stretched and shiny epidermis, with no visual signs of dehydration. <b>(C)</b> wt and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at 3 weeks. Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice showed alopecia and growth retardation. <b>(D)</b> wt and Klk5^-/-Klk7^-/-Sp5^A135X/A135X mice at 6 weeks. <b>(E)</b> Vibrissae hair obtained from P5 pups analysed by scanning electron microscopy. Klk5^-/-Klk7^-/-Sp5^A135X/A135X showed hair shaft abnormalities similar to bamboo hair; Scale bar, 30 μm. <b>(F)</b> Progression of body weight of wt, Klk5^-/-Sp5^A135X/A135X, Klk5^-/-Klk7^-/-Sp5^A135X/A135X and Klk5^-/-Klk7^-/- mice, n>5, error bars represent standard errors of mean.</p

Generation of <i>Spink5</i> mutant mice.

<p><b>(A)</b> Nucleotide and amino acid sequences of human SPINK5/LEKTI (left). The two-bp deletion 398delTG in exon5 of human SPINK5 gene results in a frame-shift and PTC (red box) as described in Raghunath et al., 2004(23). Comparison of corresponding sequences in murine Spink5/LEKTI (right) where deletion of TG nucleotides at position 402 (black underline) has the same impact as in humans. <b>(B)</b> Position of critical TG nucleotides (black underline) in the exon5 of murine Spink5 gene. TALEN-binding sites are marked with red underline, position of primers for PCR screening (F1, R1, F2, R2) are denoted. As a targeting construct, a single-stranded oligonucleotide having sequences homologous to wt DNA (blue underline) flanked desired mutation. Premature STOP codon (red box) and introduced new <i>Xba</i>I recognition sequence are depicted. <b>(C)</b> RFLP analysis of targeted mice. PCR product amplified from genomic DNA using primers F1 and R1 was digested using <i>Xba</i>I enzyme. Cleavage products of 283 and 220 bp originate from the positively targeted allele, a 503 bp fragment marks the wt allele. <b>(D)</b> Expression of Spink5 mRNA was analysed by semi-qPCR analysis in Sp5^A135X/A135X mice using primers F2 and R2. Expression of GAPDH was used as a control. <b>(E)</b> Phenotype of Sp5^A135X/A135X newborn pups. Areas of peeling skin are marked with black arrowheads.</p

MMP19^−/− mice show impaired ear swelling and inflammatory reaction in CHS.

<p>(A) Five days after abdominal sensitization with the hapten (FITC), mouse ears were challenged with FITC and ear thickness was measured after 24, 48, and 72 h. Mean values are given in mm as difference to time point 0 h. (B) Hematoxylin-eosin staining of ear sections 24 h after challenge shows reduced influx of neutrophils and eosinophils in MMP19^−/− mice. (C) Staining with anti-Ki-67 antibody revealed that proliferation of keratinocytes in MMP19-deficient mice was strongly reduced in basal and suprabasal layers. Ki-67-positive cells were counted and calculated as percentage of basal and total keratinocytes. Six images (magnification 400x) per mice were analyzed. (D) Decreased processing of IGFBP-3 in MMP19-deficient mice. Primary keratinocytes from MMP19^+/− and MMP19^−/− mice were grown for 72 h and conditioned media were analyzed for IGFBP-3 proteolysis by western blotting. The arrowhead indicates the position of intact IGFBP-3, whereas the arrow points to 30 kD IGFBP-3 proteolytic fragment. (E) Anti-CD8 staining (red) of ear sections. MMP19^−/− mice show low numbers of CD8⁺ T cells in CHS (upper panel) as well as in a T cell-independent model of inflammation, i.e. irritant dermatitis, induced by croton oil (middle panel). Ears of unsensitized mice (lower panel) exhibit low numbers of CD8⁺ cells; no difference was observed between MMP19^+/+ and MMP19^−/− mice. CHS and irritant dermatitis were carried out in four independent experiments each with 4 wild-type and 4 MMP19^−/− mice. (F) 24 h after FITC challenge ear lysates were analyzed for cytokine expression that was generally reduced in MMP19-deficient mice compared to wild-type animals. (G) Reduced levels of lymphotactin and I-TAC from three independent experiments are shown. Bars in F and G represent values of MMP19^−/− mice given as fold decrease to wild-type mice. Significant values with *p<0.05 and **p<0.01; student's t-test. Scale bars: B, 50 µm; C, 20 µm; D, 50 µm.</p

T cell population in blood of immature MMP19^−/− mice is distorted.

<p>(A) Blood samples of 3-weeks old MMP19^+/+ and MMP19^−/− mice were analyzed for monocytes, granulocytes (CD11b and Ly-6), B cells (B220), and T cell populations by flow cytometry. MMP19^−/− mice exhibit significantly reduced numbers of CD4⁺ and CD8⁺ T cells. (B) A typical dot plot analysis of CD4⁺ and CD8⁺ populations gated on CD3⁺ cells in individual mice is shown. (C) No differences in T cell subpopulations were measured in blood of adult mice (12 weeks old). CD4⁺ and CD8⁺ T cells shown in A and C are also positive for CD3. Analyses were carried out in five independent experiments with MMP19^+/+ (n = 4) and MMP19^−/− mice (n = 4). Numbers of B and T lymphocytes were analyzed by gating the lymphocyte region. Significances with *p<0.05; student's t-test.</p

Generation of MMP19^−/− mice.

<p>(A) Schematic representation of the murine <i>mmp19</i> gene and its exon/intron-organization as previously described by Mueller et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002343#pone.0002343-Mueller1" target="_blank">[7]</a>; pPNT targeting vector construct, and the resulting deleted active site locus of the mouse <i>mmp19</i> gene are depicted. The targeting construct based on the pPNT vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002343#pone.0002343-Tybulewicz1" target="_blank">[70]</a>, was generated by replacement of 1088 bp region of <i>mmp19</i> gene spanning the end of exon 3 and the whole exon 4 encoding the catalytic domain by the neomycin resistance cassette. Homologous recombination led to introduction of the PGK-Neo cassette and allowed selection of homologous recombinants. The 3′-probe used for detection of replacement events is indicated by thick bars. Also shown are restriction sites used for southern hybridization screening as well as primer binding sites used for diagnostic PCR. Screening for replacement mutants employed restriction digestion of genomic DNA with <i>Stu</i>I and <i>Eco</i>RV and southern hybridization with the described 3′-probe. (B) For screening of MMP19-deficient mice Southern blot analyses were performed: mouse DNA digested with <i>Stu</i>I and <i>Eco</i>RV was probed with the diagnostic 3′-probe. Probing led to identification of either a 7.3 kb band (wild-type allele, +/+) or a 5 kb band for the targeted allele (−/−). In heterozygous mice (+/−) both alleles are present. (C) Genotyping of targeted alleles using PCR. Wild-type alleles are detected by an 800 bp band, while PCR for the MMP19-deficient allele results in a 600 bp product. (D) Primary keratinocytes isolated from wild-type, heterozygous, and homozygous MMP19-deficient mice were analyzed for MMP19 expression by western blotting using anti-MMP19 antibodies purified against a peptide derived from the hinge region of murine MMP19, that is deteced in size of 59 kD. (E) Immunohistochemical analysis of murine skin with anti-MMP19 antibodies described above. Scale bars: 50 µm.</p