Non-Essential Role for TLR2 and Its Signaling Adaptor Mal/TIRAP in Preserving Normal Lung Architecture in Mice

<div><p>Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an <i>in vivo</i> genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in <i>Tlr4^−/−</i> mice by 6 months of age, the lungs of <i>Tlr2^−/−</i> mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in <i>Tlr4^−/−</i> mice, along with up-regulation of NADPH oxidase 3 (<i>Nox3</i>) mRNA expression. With respect to Mal, the architecture of the lungs of <i>Mal^−/−</i> mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free <i>Mal^−/−</i> mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous <i>Tlr4^−/−</i> mice. In conclusion, our data provide <i>in vivo</i> evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema.</p></div

Comparative stereological analyses of lungs from wild-type (+/+), Tlr4^−/− and Tlr2^−/− mutant mice.

<p>Data are expressed as the mean ± SEM. n = at least 5 mice per genotype. *<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 and ***<i>P</i>&lt;0.001 versus age-matched +/+ mice. Vv = volume fraction; par = parenchyma; air = air space; sep = septal tissue; Sv = surface density; S = surface area.</p

Reduced gene expression of <i>TLR4</i>, but not other TLR family members, in human emphysema tissue.

<p>Q-PCR gene expression analyses of <i>TLRs 1–6</i> were performed on human lung cDNA from emphysema (Emp) patients (n = 14) or emphysema-free (Control) individuals (n = 9). Expression data are shown following normalization for <i>18S</i> expression, and are presented from triplicate analysis as the mean ± SEM. *<i>P</i>&lt;0.05 versus emphysema-free controls.</p

Increased oxidative stress and pulmonary apoptosis in <i>Tlr4^−/−</i>, but not <i>Tlr2^−/−</i>, mice.

<p>(<b>A</b>) Protein carbonylation levels were determined by Oxyblot of lung protein lysates from 6 month old +/+, <i>Tlr4^−/−</i> and <i>Tlr2^−/−</i> mice. Each lane represents tissue from an individual mouse. Densitometric quantification of total protein carbonylation in representative samples per genotype was performed and normalized against actin protein levels present in each sample. Data are presented as the mean fold induction ± SEM for at least n = 4 per genotype. Q-PCR expression analyses of (<b>B</b>) <i>Nox2</i> and (<b>C</b>) <i>Nox3</i> were performed on lung cDNA from 6 month old mice of the indicated genotypes. Expression data are shown from at least n = 4 per genotype following normalization for <i>18S</i> expression, and are presented from triplicate analysis. (<b>D</b>) Representative photomicrographs showing TUNEL-stained cells in a cross-section of the lungs from the indicated genotypes at 6 months of age. Stereological quantification of the percentage of TUNEL-stained cells in the lungs of mice. Data are expressed as mean ± SEM, n = at least 3 mice per genotype. *<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 versus age-matched +/+ mice.</p

Alveolar air space enlargement, increased lung volume and static compliance in <i>Tlr4^−/−</i>, but not <i>Tlr2^−/−</i>, mice.

<p>(<b>A</b>) Representative methylene blue stained cross-sections and (<b>B</b>) representative H&amp;E stained cross-sections of lungs from wild-type (+/+), <i>Tlr4^−/−</i> and <i>Tlr2^−/−</i> mice aged 6 months. Arrows indicate enlarged airspaces. Scale bars = 100 µm (<b>C</b>) Lung volume per body weight of +/+ and <i>Tlr4^−/−</i> and <i>Tlr2^−/−</i> mice. (<b>D</b>) Static compliance of 6 month old +/+, <i>Tlr4^−/−</i> and <i>Tlr2^−/−</i> mice was assessed by flexiVent lung function analysis. Data are expressed as mean ± SEM, n = at least 3 mice per genotype. *<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 versus age-matched +/+ mice. (<b>E</b>) Q-PCR expression analyses of <i>Tlr4</i> was performed on lung cDNA from 6 month old +/+ and <i>Tlr2^−/−</i> mice. Expression data are shown from n = 4 per genotype following normalization for <i>18S</i> expression, and are presented as mean ± SEM from triplicate analysis.</p

No changes in the emphysematous parameters in <i>Mal^−/−</i> mice.

<p>(<b>A</b>) Representative methylene blue stained cross-sections and (<b>B</b>) representative H&amp;E stained cross-sections of lungs from <i>Mal^+/+</i> (+/+) and <i>Mal^−/−</i> mice aged 6 months. Scale bars = 100 µm (<b>C</b>) Lung volume per body weight of +/+ and <i>Mal^−/−</i> mice (<b>D</b>) Static compliance of 6 month old +/+ and <i>Mal^−/−</i> mice was assessed by flexiVent lung function analysis. Data are expressed as mean ± SEM, n = at least 3 mice per genotype. *<i>P</i>&lt;0.05 versus age-matched +/+ mice. (<b>E</b>) Q-PCR expression analyses of <i>Mal</i> was performed on lung cDNA from 6 month old +/+ and <i>Tlr4^−/−</i> mice. Expression data are shown from n = 4 per genotype per following normalization for <i>18S</i> expression, and are presented as mean ± SEM from triplicate analysis. (<b>F</b>) Q-PCR expression analysis of <i>MAL</i> was performed on human lung cDNA from emphysema (Emp) patients (n = 14) or emphysema-free (Ctl) individuals (n = 9). Expression data are shown following normalization for <i>18S</i> expression, and are presented from triplicate analysis as the mean ± SEM.</p

Slfn4 over-expression caused splenomegaly.

<p>(A) Macroscopic appearance of spleens from <i>Slfn4</i> over-expressing mice (right) and from MacBlue littermate controls (left). (B) Organ weights are expressed as percentage of body weight. Data are combined from four independent experiments and are displayed as mean + SEM.</p

Increased neutrophils and macrophages in the livers and spleens of MacBlue/UAS-<i>Slfn4</i>-V5 mice.

<p>(A) Hematoxylin and eosin staining was performed on liver paraffin sections. Arrowheads indicate aggregates of neutrophils in the liver. Liver sections from MacBlue/UAS-<i>Slfn4</i>-V5 and MacBlue littermate controls were also stained for F4/80 (brown). Arrows indicate F4/80 expressing cells surrounding aggregates of neutrophils. (B) Hematoxylin and eosin staining was also performed on spleen paraffin sections. Arrows indicate clusters of megakaryocytes. Spleen sections from MacBlue/UAS-<i>Slfn4</i>-V5 and MacBlue littermate controls were also stained for F4/80 (brown). Sections were examined using an Olympus BX-51 microscope with a DP-70 digital camera and DP controller imaging software (Olympus). RP, red pulp; WP, white pulp.</p

Increased macrophages and granulocytes in spleen from MacBlue/UAS-<i>Slfn4</i>-V5 mice.

<p>Splenocytes from MacBlue littermate controls (A) and MacBlue/UAS-<i>Slfn4</i>-V5 mice (B) were stained for the cell surface markers F4/80 (macrophage), Ly-6G (granulocyte), B220 (CD45R, B cell), and CD3ε (T cell). The samples were analysed by FACS and quadrants were set based upon isotype control profiles. Data are representative of two independent experiments.</p

Macrophage-specific expression of the UAS-<i>Slfn4</i>-V5 transgene.

<p>(A) Elements of the UAS-<i>Slfn4</i>-V5 transgene include six UAS, a kozak sequence, and the open reading frame of <i>Slfn4</i> followed by a V5-tag. Upon crossing of the UAS-<i>Slfn4</i>-V5 mouse with the MacBlue mouse, the offspring (MacBlue/UAS-<i>Slfn4</i>-V5 mice) contain the GAL4-expressing module, the GAL4-reporting module, and the UAS-<i>Slfn4</i>-V5 transgene. GAL4/VP16 protein binding to both the UAS induces the expression of ECFP and <i>Slfn4</i>-V5 specifically in cells of the myeloid lineage. (B and C) RNA from bone marrow (BM) or BMM from the offspring of four UAS-<i>Slfn4</i>-V5 founder lines (F1–F4) was extracted and cDNA was prepared. <i>Slfn4</i> mRNA levels relative to <i>hprt</i> were determined by quantitative real-time PCR. Data are combined from at least two independent experiments (mean + range) and are presented as expression relative to BM controls to enable normalization across different the transgenic lines (B), or as expression relative to <i>hprt</i> (no normalization across the transgenic lines) (C).</p