mCLCA3-deficiency in <i>S. aureus</i> infection was not compensated by regulation of other CLCA members.

<p>Lung expression mRNA levels of mClca1 to 7 were quantified by RTq-PCR. Dotted lines indicate a fold change of 0.5 and 2, respectively, as limits for valid statement of lowered and elevated parameters. Values are given as mean ± SEM (n = 8 each group). Ct, cycle treshold.</p

Pulmonary neutrophils and lymphocytes were significantly decreased in mCLCA3-deficient mice 24 hours after infection.

<p>(A, B) At 12 hours and 24 hours after infection, leukocytes in bronchoalveolar lavage fluid (BALF) were manually counted and subpopulations were analyzed by fluorescence-activated cell sorter (FACS). Values are given as mean ± SEM (n = 8 each group). ^#p<0.05, ^##p<0.01, ^###p<0.001, ^####p<0.0001 versus PBS controls. *p<0.05 as indicated. (C) Cytospins from BALF 24 hours post infection were obtained by centrifugation and BALF cell suspension was subsequently stained with May-Grünwald Giemsa. Leukocyte numbers were reduced in BALF from mClca3^−/− mice (n = 8 each group). <i>Bar,</i> 50 µm.</p

mCLCA3-deficiency had no influence on mucus cell number and mucin expression in infected mice.

<p>(A) Mucus cells were quantified by calculating the percentage of periodic acid Schiff (PAS)-positive cells per mm basement membrane. (B) No differences in number or distribution of mucus cells were observed in <i>S. aureus</i> infected or uninfected mice. (C, D) Expression levels of Muc5ac, Muc5b and Muc2 were quantified at indicated time points in trachea and lung by RTq-PCR. Dotted lines indicate fold change of 0.5 and 2, respectively, as limit for valid statement of lowered and elevated parameters. Values are given as mean ± SEM (n = 4 each group (A, B), n = 8 each group (C, D)). ^#p<0.05, ^##p<0.01, ^###p<0.001, versus the PBS control group. <i>Bar</i> (B), 20 µm.</p

mCLCA3-deficiency led to reduced protein quantities in BALF during infection without altered pulmonary vascular permeability.

<p>(A) Total protein of BALF was examined at indicated time points by BCA-assay. (B) Mouse albumin (MA) of transnasally infected or uninfected mice was measured by ELISA in BALF and plasma. Pulmonary vascular permeability was calculated from the MA BALF/plasma ratio. Values are given as mean ± SEM (n = 8 each group). ^#p<0.05, ^##p<0.01 versus the PBS control group. *p<0.05 as indicated.</p

mCLCA3 had no impact on severity or expansion of lung inflammation.

<p>(A) 24 hours after infection, left lungs of <i>Staphylococcus aureus</i> infected mice were used for quantification of lung lesions by Cavalieri principle. (B) Percentages of estimated lung lesion volumes and total lung volumes of the left lungs were calculated. Values are given as mean ± SEM (n = 4 each group).</p

No genotype differences in lung inflammation, lesion distribution or bacterial loads were observed during infection.

<p>Mice were transnasally infected with 5×10⁷<i>Staphylococcus aureus</i> Newman and killed at indicated time points. (A) Macroscopic examination revealed deeply red consolidated areas in the infected lungs (arrowhead) in contrast to PBS controls which behaved virtually identical. (B) Subsequently, lungs were fixed, embedded in paraffin and stained with hematoxylin and eosin for histopathological analyses. (C) Evidence of bacteria was assessed by immunohistochemistry with anti-<i>Staphylococcus aureus</i> antibody. Uninfected animals (left panel) served as negative controls. <i>Brown</i>, 3,3′-diaminobenzidine; <i>blue</i>, hematoxylin counterstain. (D) The total lung area affected by inflammation and (E) a lung inflammation score were determined. Values are given as mean ± SEM (n = 4 each group). ^#p<0.05 versus the PBS control group. <i>Bar</i> (B), 100 µm. <i>Bar</i> (C), 20 µm.</p

Lung Scoring Parameters.

<p>Lung Scoring Parameters.</p

Chemokine CXCL-1 and cytokine IL-17 were significantly decreased in BALF of infected mCLCA3-deficient mice.

<p>(A, B) Protein levels of cytokines were measured at 12 hours and 24 hours post infection by multiplex assay technique (IL-1β, IL-6, IL-10, IL-12p40, IL-13, CXCL-1, MCP-1, RANTES, TNF-α, GM-CSF) or by ELISA (IL-17) in BALF. (C) Expression levels of Cxcl-1, Cxcl-2 and Il-17 were quantified by RTq-PCR in the lungs. Dotted line indicates a fold change of 2 as limit for valid statement of elevated parameters. Values are given as mean ± SEM (n = 6 to 8 each group). ^#p<0.05, ^##p<0.01, ^###p<0.001, ^####p<0.0001 versus PBS control group. *p<0.05 as indicated.</p

β5i/LMP7^-/- mice exhibit a diminished expression of opsonizing molecules in lung, liver and macrophages.

<p>(A) Gene expression analysis of lungs, 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, performed by RT and Real Time PCR (each group with n = 5–6; statistical analysis by Mann Whitney U Test. *p<0.05). (B) Gene expression analysis of BMM co-cultivated with D39Δcps (MOI 10) for 4 h, performed by RT and Real Time PCR (each group with n = 4, illustrated experiment is representative for 4 individual replicates; showing mean± SD; statistical analysis by student’s t-test **p<0.01). (C) Gene expression analysis of liver 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, performed by RT and Real Time PCR (each group with n = 12–14; statistical analysis by Mann Whitney U Test. *p<0.05; **p<0.01).</p

Both immuno- and standard subunits of the proteasome are constitutively expressed in leukocytes and liver.

<p>(A) Analysis of proteasome catalytic β-subunits expression on protein level in liver, 48 h after transnasal application of 5×10⁶ CFU PN36/mouse, performed by immuno-blot. (B) Analysis of proteasome catalytic β-subunits expression on protein level in BMM performed by immuno-blot (each group with n = 3). (C) 2D gel electrophoresis of purified 20S proteasomes, isolated from WT BMM. Proteasome subunits were visualized by coomassie stain. (D) Determination of β-subunits, incorporated in BMM 20S, detected by immuno-blot.</p