CD44 as a novel target for treatment of staphylococcal enterotoxin B-induced acute inflammatory lung injury

Clinical Immunology (2012) 144, 41–52

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

CD44 as a novel target for treatment of staphylococcal enterotoxin B-induced acute inflammatory lung injury Jingping Sun, Gabriela P. Law, Christy C. Bridges, Robert J. McKallip ⁎ Division of Basic Medical Sciences, Mercer University School of Medicine, USA

Received 7 April 2012; accepted with revision 3 May 2012 Available online 11 May 2012

KEYWORDS Lung inflammation; Staphylococcal enterotoxin B; CD44

Abstract Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In the current study, we investigated the role of CD44 in ALI/ARDS. Intranasal exposure of CD44 wildtype mice to SEB led to a significant increase in the expression of CD44 on lung mononuclear cells. CD44 knockout mice developed significantly reduced SEB-induced ALI/ARDS, through reduced inflammatory cytokine production and reduced lung inflammatory cells, compared to similarly treated CD44 wild-type mice. Mechanistically, deletion of CD44 altered SEB-induced cytokine production in the lungs and reduced the ability of SEB-exposed leukocytes to bind to lung epithelial cells. Finally, treatment of SEB-exposed mice with anti-CD44 mAbs led to significant reduction in vascular permeability, reduction in cytokine production, and prevented inflammatory cell infiltration in the lungs. Together, these results suggest the possibility of targeting CD44 for the treatment of SEB-induced ALI/ARDS. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) and more recently community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are major emerging health threats with a mortality rate of approximately 20%. In fact it is estimated that MRSA infections will result in more fatalities than those related to HIV. Those infections resulting in pneumonia or toxic shock ⁎ Corresponding author at: Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College St., Macon, GA 31207, USA. Fax: + 1 478 301 5487. E-mail address: [email protected] (R.J. McKallip). 1521-6616/$ - see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2012.05.001

syndrome/sepsis have high mortality rates of approximately 32% and 56%, respectively [1]. S. aureus infection-related diseases are often a consequence of exposure to superantigens such as SEB which leads to clonal activation of up to 40% of naïve T and possibly NKT cells. Activation of these lymphocytes leads to a cytokine storm characterized by the release of large quantities of pro-inflammatory cytokines such as IL-2, IL4, IL-6 and IFN-γ [2] which can lead to endothelial cell injury, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and vascular collapse (shock) [3]. In addition, SEB is listed as a potential bioweapon [4]. Currently, there are no effective treatments for the resulting inflammatory response. CD44 is a widely distributed cell surface glycoprotein expressed by a number of lymphoid and non-lymphoid

42 tissues. CD44 exists as a number of isoforms, which differ in their molecular weights, ranging from 85 to 260 kDa [5,6]. Evidence suggests that CD44 can be directly involved in a number of processes important in the development of lung inflammation. For example, lymphocytes can be triggered to proliferate by binding of mAbs specific for CD44 [7]. This proliferative response was comparable to that seen when the cells were stimulated with anti-CD3 mAbs and was IL-2 dependent. In addition to T lymphocytes, NK cell activation is also affected by CD44 binding as CD44 significantly increased NK cell lysis of NK-sensitive targets [8]. CD44 has also been shown to play an important role in the inflammatory response by acting as an adhesion molecule and binding to hyaluronic acid (HA). Interactions between CD44 and HA have been implicated in the normal function of the immune system as well as in a number of human disease models. For example, CD44 interacting with HA has been reported to play an important role in the recruitment of lymphocytes to sites of inflammation [9,10]. In addition, CD44 and HA have been shown to be important for the migration of neutrophils as well as macrophages in response to an inflammatory stimulus [11–13]. CD44 has been reported to act as an adhesion molecule by binding to E-selectin and L-selectin [14–17]. Furthermore, CD44 has been reported to play a direct role in the production of a number of proinflammatory molecules including IL-1β, IL-2, IL-6, IL-10, IFN-γ, and TGF-β [18–20]. CD44 has also been reported to play a role in lung inflammation. For example, we reported a role of CD44 in the development of IL-2-induced vascular leak syndrome [3,21]. Additionally, CD44 has been shown to influence lung damage in other models. For example, CD44 was shown to play an important role in the development of lung injury following abdominal sepsis, exposure to ozone, as well as a role in the development of antigen-induced asthma [22–24]. In contrast, other reports suggest a potential protective role of CD44 in immune-mediated lung injury. For example, CD44 expression was shown to reduce the severity of LPS-induced pulmonary inflammation [25]. Taken together, there is a large body of evidence suggesting an important role of CD44 in the inflammatory response. However, to date, the relevance of CD44 in the development of SEB-induced lung injury has not been reported. In the current study, we tested the hypothesis that SEB activation leads to increased expression of CD44 which plays an important role in the development of SEB-induced ALI/ ARDS. Furthermore we examined the possibility of targeting CD44 in the treatment of SEB-induced ALI/ARDS. Knowledge gained from this study will advance our understanding of the role of CD44 in SEB-mediated vascular damage and may ultimately lead to significantly improved treatment of symptoms associated with SEB exposure and/or MRSA infections.

2. Materials and methods 2.1. Mice Adult female C57BL/6 (CD44WT) mice were purchased from the National Institutes of Health (Bethesda, MD). CD44 knock out (CD44KO) mice were purchased from the Jackson Laboratory (Bar Harbor, MA) and bred in our animal facilities

J. Sun et al. and screened for the CD44 mutation. All these strains were on C57BL/6 background.

2.2. Cell lines The mouse vascular epithelial cell (MAEC) cell line was obtained from RIKEN BioResource Center (Tsukuba, Japan). This cell line has been reported to retain many of the important morphologic biological characteristics of normal epithelial cell lines making it a useful tool for studying interactions between SEB-activated leukocytes with epithelial cell lines in vitro [26]. The growth medium employed in this study was M199 medium (Sigma) with 5 ng/ml of recombinant vascular endothelial growth factor (VEGF; Sigma), Hepes (Invitrogen, Carlsbad, Calif., USA), heparin sodium and 5% FBS [26].

2.3. Quantification of vascular permeability Vascular leakiness was studied by measuring the extravasation of Evan's blue, which when given i.v. binds to plasma proteins, particularly albumin, and following extravasation can be detected in various organs as described previously [27]. Vascular leak was induced by injection of SEB, as previously described [28,29]. Briefly, groups of five mice were injected i.n. with SEB (20 ng) or PBS. The mice were exposed to SEB for 6–48 h. Two hours prior to harvesting the lungs the mice were injected i.v. with 0.1 ml of 1% Evan's blue in PBS. After 2 h the mice were exsanguinated under anesthesia, and the heart was perfused with heparin in PBS as described previously [30]. The lungs were harvested and placed in formamide at 37 °C overnight. The Evan's blue in the organs was quantified by measuring the absorbance of the supernatant at 650 nm with a spectrophotometer. In experiments examining the effect of anti-CD44 mAbs on SEBinduced vascular permeability, mice were treated with isotype control mAbs or anti-CD44 mAbs (IM7, 100 μg/ mouse). The vascular permeability seen in SEB-exposed mice was expressed as percent increase in extravasation when compared with that of PBS-treated controls and was calculated as: [(optical density of dye in the lungs of SEB − exposed mice) − (optical density of dye in the lungs of PBStreated controls)] / (optical density of dye in the lungs of PBS-treated control) × 100. Each mouse was individually analyzed for vascular permeability, and data from five mice were expressed as mean ± SEM percent increase in vascular permeability in SEB-exposed mice when compared to that seen in PBS-treated controls [28,29].

2.4. Histological analysis Groups of 3 mice were exposed to SEB or PBS as described earlier, and 24 h later, the lungs were fixed in 10% formalin solution. The organs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E), as described [28,29]. Lung sections were assigned a subjective histological score as described elsewhere [31,32]. Briefly, lung sections were assigned a score from 0 to 3. A value of zero was assigned to sections where there was no detectable inflammation, a score of 1 was assigned to section where occasional cuffing with inflammatory cells was observed, a

CD44 for the treatment of SEBnduced ALI score of 2 was assigned to sections where most bronchi or vessels were surrounded by a layer of one to five cell thick of inflammatory cells, a value of 3 was assigned to sections where most bronchi or vessels were surrounded by a thick layer of inflammatory cells (greater than five cells thick) and a value of 4 was assigned to sections where most bronchi or vessels were surrounded by a thick layer of inflammatory cells (greater than five cells thick) and infiltrate was observed in the alveoli.

2.5. Quantification and identification of lung-infiltrating leukocytes Mice were exposed to SEB or PBS as described earlier; 24 h later the lungs were harvested and prepared into a single cell suspension using a laboratory homogenizer. The cells were washed and mononuclear cells were layered on histopaque and isolated by gradient centrifugation. The cells were then quantified by Trypan blue dye exclusion and the phenotype of the cell was determined by flow cytometric analysis. For flow cytometric analysis a number of antibodies conjugated to fluorescent probes were used. T cells (NK1.1 −CD3 +), NK cells (NK1.1 +CD3 −), and NKT cells (NK1.1 +CD3 +) were identified using anti-CD3 and anti-NK1.1 mAbs. For macrophage, Mac-3 antibodies were used. Neutrophils were characterized using anti-CD45 and antiGr-1 antibodies (CD45 +Gr-1 high) [33]. The cells (10,000) were analyzed by flow cytometry using a BD FACSAria II cell sorter (BD Bioscience, San Jose, CA, USA). The total cell number for individual mononuclear cell populations was calculated using the following equations: (cell number of specific mononuclear cell population = percentage of specific cell population × total number of mononuclear cells isolated from the lung).

2.6. RNA isolation, RT-PCR and real-time RT-PCR analysis Total RNA was isolated from single cell suspension of splenocytes using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA concentration and integrity were determined spectrophotometrically. cDNA was synthesized by reverse transcription of 50 ng total RNA using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Carlsbad, California). Real-time PCR was performed using a SYBR Green PCR kit (Applied Biosystems, Carlsbad, CA). Amplications were performed and monitored using an ABI 7300 realtime PCR system (Applied Biosystems, Carlsbad, CA). The gene-specific primers for β-actin have been previously described [30]. In addition the following primers were used: IL-2 primers 5′-TGATGGACCTACAGGAGCTCCTGAG-3′ and 5′-GAGTCAAATCCAGAACATGCCG CAG-3′, IL-4 primers 5′-CCAGCTAGTTGTCATCCTGCTCTT CTTTCTCG-3′ and 5′-CA GTGATGTGGACTTGG ACTCATTCATGGTGC-3′, IL-6 primers 5′-ATGAAGTTCCTCTCTGCAAGAGACT-3′ and 5′-CACTAGGTT TGCCGAGTAGATCTC-3′ and IFN-γ primers 5′-TGCATC TTG GCTTTGCAGCTCTTCCTCATGGC-3′ and 5′-TGGACCTGTGGGT TGTTG ACCTCAAACTTGGC-3′. The threshold cycle (ΔCT) method was used for relative quantification of gene transcription in relation to expression of the internal standard β-actin. Fold changes of mRNA levels in SEB-

43 stimulated mononuclear cells relative to unstimulated cells were determined using the 2 − ΔΔCt method [34].

2.7. Bead array analysis of cytokine levels Cytokine assessment was carried out using the BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17Cytokine Kit (BD Bioscience, San Jose, CA, USA) for simultaneous detection of seven cytokines (IL-2, IL-4, IL-6, IL-10, IL17a, TNF-α and IFN-γ) in BALF of PBS or SEB exposed mice. Cytokines were determined in the BALF samples according to the manufacturer's instructions. Briefly, test samples and PE detection antibody were incubated with capture beads for 3 h in the dark at room temperature. After which, all unbound antibodies were washed and resuspended in 300 μl PBS. The cytokine levels were analyzed using a BD FACSAriaII cell sorter. Cytokine levels were calculated using the FCAP Array Software v3.0 (BD Bioscience, San Jose, CA, USA).

2.8. Cell adhesion assay MAECs were plated at 1 × 10 4 /well and cultured at 37 °C overnight in a 96-well flat-bottomed plate. Naïve or SEBexposed CD44WT and CD44KO splenocytes were labeled with CFSE (carboxyfluorescein diacetate succinimidyl ester, Molecular Probes, Eugene, OR). The spleen cells were washed and added to wells containing MAEC cultures and incubated for 2 h at 37 °C. Splenocytes not adhering to MAECs were removed by gently washing the cultures with medium. Washing was performed by removing the initial media followed by two washing steps, which consisted of gentle pipetting with 100 μl of media. The percentage of adherent cells was quantified by measuring the level of fluorescence remaining following washing of the cells compared to the fluorescence in non-washed wells (total fluorescence). Fluorescence was quantified using a Tecan Infinite M1000 multimode plate reader (Männedorf, Switzerland). The percentage of splenocytes adhering to the MAEC was determined using the following formula: (fluorescence of adhering cells / total fluorescence) × 100%.

2.9. Proliferation assay The spleens were harvested from euthanized CD44WT and CD44KO mice and placed into 10 ml of RPMI 1640 media (Gibco Laboratories, Grand Island, NY) supplemented with 5% FCS, 10 mM HEPES, 1 mM glutamine, 40 μg/ml gentamicin sulfate, and 50 μM 2-mercaptoethanol, referred to as complete media. The spleens were prepared into a single cell suspension using a laboratory homogenizer, washed twice, and adjusted to 5 × 10 6/ml in complete media. The splenocytes (5 × 10 5 in 100 μl/well) were cultured in 96-well flat-bottomed plates were left unstimulated or stimulated with 2 μg/ml SEB for 24 or 48 h. Cell viability was assayed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay absorbance.

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2.10. Detection of apoptosis The spleens were aseptically harvested from CD44 WT and CD44 KO mice and prepared into a single cell suspension as described above. The splenocytes were adjusted to 5 × 10 6/ ml in complete medium and added to 96 well plates (100 μl/ well) unstimulated or stimulated with SEB (20 μg/ml) for 24 h. Next, the splenocytes were harvested and analyzed for apoptosis using the TUNEL methods [35]. To detect apoptosis using the TUNEL method, the cells were washed twice with PBS and fixed with 4% p-formaldehyde for 30 min at room temperature. The cells were next washed with PBS, permeabilized on ice for 2 min and incubated with FITC-dUTP and TdT (Promega, Madison WI) for 1 h at 37 °C and 5% CO2 [35]. The levels of apoptosis were determined by measuring the fluorescence of the cells by flow cytometric analysis. Ten thousand cells were analyzed per sample

2.11. Statistical analysis ANOVA was used to determine statistical significance and p ≤ 0.05 was considered to be statistically significant.

3. Results 3.1. SEB exposure leads to increased CD44 expression in lung mononuclear cells To determine whether SEB exposure had an effect on the expression levels of CD44 in lung mononuclear cells, C57BL/6 mice were exposed i.n. to SEB (5 ng/50 μl PBS). Lung mononuclear cells were isolated 24 h later and stained with Cy5-conjugated anti-CD44 mAbs (Fig. 1A). CD44 levels were determined by flow cytometric analysis and revealed that SEB exposure led to a significant increase in CD44 expression increasing from a mean fluorescence intensity (MFI) of 272 in the PBS-treated mice to a MFI of 480 in the SEB-exposed mice. In addition, SEB exposure resulted in an increase in the CD44 high expressing population. More specifically, we demonstrated an increase from 30% CD44 high cells in the PBStreated mice to 61% in the SEB-treated mice. Finally, the effect of SEB on CD44 expression on individual cell populations was determined and demonstrated an increase in CD44 expression on all mononuclear cells assayed including; T cells, NK cells, NKT cells, macrophage, and neutrophils (Fig. 1B).

3.2. CD44KO mice are resistant to SEB-induced vascular permeability Next we examined whether deletion of CD44 had any influence on the development of SEB-induced ALI/ARDS using lung permeability as a measure of compromised lung integrity. To this end, CD44WT and CD44KO mice were exposed to PBS or SEB (20 ng/50 μl PBS i.n.). The level of vascular permeability was determined 24, 48, and 72 h following SEB exposure. The level of vascular leak in SEBexposed mice was compared to the level of vascular leak in PBS-exposed mice and depicted as the percent increase over

Figure 1 SEB exposure leads to increased CD44 expression in lung mononuclear cells. CD44WT mice were exposed i.n. to PBS or SEB for 24 h. After which, the lung mononuclear cells were collected and stained with APC-labeled anti-CD44mAb (solid line) or APC-labeled isotype control mAb (dotted line) (A). The data depict the mean fluorescence intensity (MFI) from a representative experiment. In addition, mononuclear cells isolated from PBS or SEB-exposed CD44WT were stained with FITC-conjugated anti-CD3 mAbs, PE-conjugated antiNK1.1 mAbs, and Cy-5-conjugated anti-CD44 mAbs. In addition, cells were stained with FITC-conjugated anti-Gr-1 mAbs, PE-conjugated anti-Mac-3 mAbs, and Cy5-conjugated anti-CD44 mAbs. CD3 + , NK1.1 − (T cells), CD3 − NK1.1 + (NK cells), CD3 + NK1.1 + (NKT), Gr-1 + (neutrophils), and Mac-3 + (macrophage) cells were gated, and the level of CD44 expression on these populations was determined by FACS analysis (B). The data are depicted as mean fluorescence intensity (MFI) ± SD from 3 individual experiments. More than 1000 events were analyzed per gated population. Asterisks indicate statistically significant difference when compared to the PBS-exposed CD44WT controls, p ≤ 0.05.

leak in PBS-exposed mice [3]. The results showed that following 24, 48, and 72 h exposure to SEB there was a significant increase in vascular permeability in the CD44WT mice. However, CD44KO mice were significantly resistant to SEB-induced vascular permeability at all time points tested (Fig. 2).

CD44 for the treatment of SEBnduced ALI

45 mononuclear cells characterized. Finally, we examined the effect of CD44 deletion on SEB-induced changes in BALF cell populations. The results were similar to those seen when examining the lung infiltrating cells in that SEB exposure led to a significant increase in total BALF cell number (Fig. 3E) and specifically an increase in T cell, NK cells, NKT cells, macrophage, and neutrophils in CD44WT mice (Fig. 3F). In contrast, exposure of CD44KO mice to SEB led to significantly fewer leukocytes in the BALF (Fig. 3E). Similar to what was seen in the lung infiltrating cells, the reduction was not specific for one type of leukocyte (Fig. 3F). Together, these results provide preliminary evidence that CD44 plays a significant role in the increase in lung mononuclear cells following SEB exposure.

Figure 2 CD44KO mice are resistant to SEB-induced vascular permeability. CD44WT and CD44KO mice were exposed i.n. to PBS or SEB (20 μg). Lung permeability was determined 24, 48, and 72 h later by quantifying the extravasation of Evan's blue dye in the lungs as described in Materials and methods. The vertical bars represent the percent increase in vascular permeability ± SD following SEB exposure compared with that in the PBS-exposed controls. Asterisks indicate statistically significant difference when compared to the SEB-exposed CD44WT mice, p ≤ 0.05.

3.3. CD44KO mice show decreased inflammatory cell number in the lungs following exposure to SEB We examined the possibility that the reduced vascular leak seen in the CD44KO mice was influenced by an effect of CD44 on SEB-induced increase in lung inflammatory cells. To this end, CD44WT and CD44KO mice were exposed to PBS or SEB (20 μg/50 μl PBS i.n.). The lungs were harvested 24 h later, fixed, and stained with H&E (Fig. 3A) and assigned a histological score (Fig. 3B). The results show that in SEBexposed CD44WT mice, the number of mononuclear cells and histological score were significantly elevated following 24 h exposure. In comparison, little increase in the number of mononuclear cells was observed and the histological score was significantly reduced in the CD44KO mice following exposure to SEB. To confirm these results, additional experiments were performed to directly assess the effect of CD44 deletion on the SEB-induced increase in lung mononuclear cells. CD44WT and CD44KO mice were exposed to PBS or SEB (20 μg/50 μl PBS i.n.). Lungs were harvested 24 h later and the infiltrating mononuclear cells were harvested and counted. The results demonstrated that SEB exposure led to a significant increase in the number of mononuclear cells infiltrating the lungs of CD44WT mice. In contrast, the increase in mononuclear cells following SEB exposure, although significantly increased compared to PBStreated mice, was significantly reduced in CD44KO mice (Fig. 3C). Phenotypic characterization of the lung mononuclear cell populations demonstrated that SEB-exposure led to a significant increase in T cells, NK cells, NKT cells, macrophages, and neutrophils in CD44WT mice (Fig. 3D). Deletion of CD44 led to significantly reduced increases in all

3.4. Targeted deletion of CD44 leads to reduced expression of inflammatory cytokines Exposure to bacterial superantigens such as SEB leads to massive activation of lymphocytes and a subsequent cytokine storm which plays an important role in the development of ALI/ARDS [36,37]. In the current set of experiments we explored the potential role of CD44 expression in SEBinduced cytokine levels in the lung. To this end, CD44WT and CD44KO mice were exposed to either PBS or SEB (20 μg/50 μl PBS i.n.). BALF and whole lungs were harvested 24 h later, and cytokine protein levels were determined by cytokine bead array (Fig. 4A) and mRNA levels were determined by real-time RT-PCR (Fig. 4B). The results demonstrated that exposure of CD44WT mice to SEB led to increased expression of a number of cytokines, including IL-2, IL-4, IL-6, and IFNγ, reported to play a role in ALI/ARDS (Figs. 4A and B). In comparison, SEB exposure of CD44KO mice led to a significant reduction in the SEB-induced increase in inflammatory cytokine production.

3.5. Deletion of CD44 does not affect the proliferative response, expansion of Vβ8 + cells or the induction of apoptosis in mouse splenocytes following exposure to SEB Preliminary experiments were conducted to explore the possible mechanism associated with the reduced induction of ALI/ARDS in CD44KO mice following exposure to SEB. To this end, we examined whether deletion of CD44 had any effect on the ability of splenocytes to become activated or undergo apoptosis following exposure to SEB. More specifically, splenocytes from CD44WT and CD44KO mice were stimulated with SEB (2 μg/ml) or medium alone for 24 or 48 h. The proliferative response was examined using a MTT assay and by examining the expansion of Vβ8 + lymphocytes by flow cytometry. The results demonstrated that splenocytes from CD44KO mice were activated at a similar level when compared to splenocytes from CD44WT mice (Fig. 5A) and that there was a similar increase in Vβ8 + cells in both groups (Fig. 5B). The effect on SEB-induced apoptosis was determined using TUNEL staining and demonstrated that deletion of CD44 did not significantly alter induction of apoptosis following exposure to SEB (Fig. 5C).

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Figure 3 CD44KO mice show decreased inflammatory cell number in the lungs following exposure to SEB. CD44WT and CD44KO mice were exposed i.n. to PBS or SEB (20 μg). The effect of SEB exposure on lung mononuclear cell number was determined 24 h later by H&E (A) and histological scoring (B) as well as direct enumeration by Trypan blue dye exclusion following mononuclear cell isolation from lungs (C). Phenotypic characterization of the mononuclear cell populations was performed by staining the isolated mononuclear cells with FITC-conjugated anti-CD3 mAbs, PE-conjugated anti-NK1.1 mAbs, FITC-conjugated anti-Gr-1 mAbs, PEconjugated anti-CD45, or PE-conjugated anti-Mac-3 mAbs. The total number of CD3 +, NK1.1 − (T cells), CD3 − NK1.1 + (NK cells), CD3 + NK1.1 + (NKT), CD45 +Gr-1 high (neutrophils), and Mac-3 + (macrophage) cells was determined as described in Materials and methods (C). The data depicted are mean total cell number ± SD from a representative experiment. More than 1000 events were analyzed per gated population. Asterisks indicate statistically significant difference when compared to the PBS-exposed controls, # indicates statistically significant difference when compared to SEB-exposed CD44WT mice p ≤ 0.05.

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Figure 4 Targeted deletion of CD44 leads to a reduction of SEB-induced inflammatory cytokines. CD44WT and CD44KO mice were exposed i.n. to PBS or SEB (20 μg). The levels of lung cytokine mRNA and protein levels were determined 24 h later. The protein levels of cytokines in BALF were determined using a cytokine bead array (A). Cytokine mRNA levels in whole lung extracts were determined by real-time RT-PCR (B). Asterisks indicate statistically significant difference when compared to the cytokine levels from SEB-exposed CD44WT mice, p ≤ 0.05.

3.6. Targeted deletion of CD44 leads to reduced adhesion of SEB-activated leukocytes to epithelial cells CD44 has been reported to play an important role in the adhesion of effector cells to target cells either directly as an adhesion molecule or through its influence on cytokine production [3,19,30,38–41]. Therefore, we examined the possibility that the absence of CD44 on SEB-exposed splenocytes leads to a reduction in their adherence to mouse vascular epithelial cells (MAECs). To this end, the effect of CD44 expression on the adherence of SEB-exposed splenocytes to MAECs was examined using an adhesion assay. The results showed that, compared to naïve splenocytes from CD44WT mice, there was a significant increase in adhesion between MAECs and SEB-exposed splenocytes from CD44 WT mice (Fig. 6). In contrast, compared to naïve splenocytes from CD44KO mice, there was no significant increase in the binding of SEB-activated CD44KO splenocytes to MAECs. These results suggest that CD44 plays an important role in SEB-induced adherence of mononuclear

Figure 5 Deletion of CD44 does not affect the proliferative response, expansion of Vβ8 + cells or the induction of apoptosis in mouse splenocytes following exposure to SEB. Splenocytes from CD44WT and CD44KO mice were cultured with SEB (2 μg/ ml) or in medium alone. The proliferative response was measured at 24 h and 48 h using the MTT test. The data represent the mean O.D. from triplicate cultures (A). Asterisk indicates statistically significant difference when compared to O.D. of medium treated spleen cells. In addition, the effect of CD44 deletion on the expansion of Vβ8 + T cells was assessed at 24 h by flow cytometric analysis (B). The effect of CD44 deletion on SEB-induced apoptosis was analyzed 24 h following SEB exposure by TUNEL assay (C).

cells to epithelial cells, which may influence their ability to migrate into the lungs.

3.7. Treatment with anti-CD44 mAbs protects mice from SEB-induced lung permeability Next, we explored the possibility of targeting CD44 for the treatment of SEB-induced vascular leak. To this end, CD44WT

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J. Sun et al. neutrophils. In contrast, treatment with anti-CD44 mAbs led to a significant reduction in the SEB-induced increase in the total number of lung mononuclear cells. The effect was not specific for any mononuclear cell population as T cells, NK cell, NKT cell, macrophage and neutrophils were all reduced compared to isotype control mAb-treated mice.

3.8. Treatment with anti-CD44 mAbs leads to a reduction in SEB-induced inflammatory cytokine levels in the lungs

Figure 6 Targeted deletion of CD44 leads to reduced adhesion of SEB-activated leukocytes to epithelial cells. CFSElabeled SEB-exposed leukocytes from CD44WT and CD44KO mice were cultured for 2 h at 37 °C in plates containing adherent MAECs. LAK cells not adhering to MAEC were removed by gently washing the cultures with medium. The percentage of adherent cells was quantified by measuring the level of fluorescence remaining following washing of the cells compared to the fluorescence in non-washed wells (total fluorescence). Asterisk indicates statistically significant difference when compared with the unstimulated leukocytes, # indicates statistically significant difference when compared to SEB-exposed CD44WT leukocytes, p ≤ 0.05.

mice were exposed to PBS or SEB. Groups of mice were then treated with anti-CD44 mAbs (50 μg i.p.) or isotype control mAbs. The level of vascular permeability was assayed 24, 48, and 72 h following SEB exposure by determining the levels of Evan's blue dye in the left lobe of the lungs. The results demonstrated that as early as 24 h following SEB exposure there was a significant increase in the levels of vascular leak compared to PBS-treated control mice. Interestingly, treatment of mice with anti-CD44 mAbs led to a significant reduction in SEB-induced vascular permeability suggesting that targeting CD44 with mAbs may confer significant protection against SEBinduced vascular permeability (Fig. 7A). We further examined the effects of anti-CD44 mAb treatment on SEB-induced inflammatory cell migration into the lungs. Mice were exposed to SEB and treated with either anti-CD44 mAbs or isotype control mAbs, as described above. The effects of anti-CD44 mAbs on mononuclear cell infiltration were assessed 24 h later by harvesting lungs from control and experimental animals and staining them with H&E (Fig. 7B) and assigning a histological score (Fig. 7C). The results demonstrated that exposure to SEB led to a significant increase in lung mononuclear cells and an increase in histological score; while mice treated with antiCD44 mAbs demonstrated significantly fewer infiltrating mononuclear cells and a reduced histological score compared to isotype control treated mice. The effect of antiCD44 mAbs was further confirmed in experiments examining the total cell number and phenotype of lung mononuclear cells (Figs. 7D and E, respectively). Exposure of isotype control mAb-treated mice to SEB led to a significant increase in the total number of mononuclear cells, characterized by an increase in T cells, NK cell, NKT cells, macrophage, and

In previous experiments we demonstrated that targeted deletion of CD44 led to a significant reduction in SEBinduced cytokine production in the lungs. In the current set of experiments we explored the potential use of anti-CD44 mAbs to reduce SEB-induced cytokine levels in the lung. To this end, CD44WT mice were exposed to either PBS or SEB (20 μg/50 μl PBS i.n.). Groups of mice then received treatment with either isotype control mAbs or anti-CD44 mAbs (50 μg i.p.). BALF and whole lungs were harvested 24 h later, and cytokine protein levels in the BALF were determined by cytokine bead array (Fig. 8A) and whole lung extract mRNA levels were determined by real-time RTPCR (Fig. 8B). The results demonstrated that exposure of isotype mAb-treated CD44 WT mice to SEB led to increased expression of a number of cytokines, including IL-2, IL-4, IL-6, and IFN-γ, reported to play a role in ALI/ARDS. In comparison, treatment of CD44WT mice with anti-CD44mAbs led to a significant reduction in the SEB-induced increase in inflammatory cytokine production.

4. Discussion Exposure to bacterial superantigens, such as SEB, can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Currently, there are no effective treatments for the resulting inflammatory response. Results from this study demonstrate an important role of CD44 in the development of SEB-induced ARDS/ALI. Specifically, we show that following SEB exposure, lung mononuclear cells have elevated expression of CD44. Furthermore, we show that targeted deletion of CD44 or use of anti-CD44 mAbs led to significantly attenuated response to SEB in the lungs. This was characterized by reduced lung permeability, reduced mononuclear cells, and a reduced cytokine production. Together, this study suggests that CD44 might be a novel target for the treatment or prevention of SEB-induced lung injury. Prior to this report, other studies have suggested a potential role of CD44 in regulating the inflammatory response in the lungs. For example, we demonstrated that deletion of CD44 led to protection from IL-2-induced vascular permeability [3]. In separate studies, we demonstrated that targeting CD44 using hyaluronic acid or HA binding protein led to protection from IL-2 induced vascular damage [21]. Additional studies examining the role of CD44 in experimental asthma demonstrated that targeting CD44 with mAbs led to reduced asthma by preventing lymphocyte and eosinophil accumulation in the lungs and by inhibiting the levels of Th2 cytokines in BALF [22]. Furthermore, deletion of CD44 was shown to be protective in a LPS-

CD44 for the treatment of SEBnduced ALI

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Figure 7 Treatment with anti-CD44 mAbs protects mice from SEB-induced lung permeability. The effect of anti-CD44 mAbs on SEB-inducedvascular permeability in vivo was determined by exposing mice to SEB, as described in the Material and methods section, and treating the mice with anti-CD44 mAbs (50 μg/mouse i.p.) or isotype control mAbs (50 μg/mouse i.p.) immediately following SEB exposure. Vascular permeability was determined as described Materials and methods (A). Asterisk indicates statistically significant difference when compared to the controls, p ≤ 0.05. The effect of anti-CD44 mAbs on the SEB-induced increase in lung mononuclear cells was determined by staining lung sections with H&E (B), by histological scoring (C), and by direct enumeration by Trypan blue dye exclusion following mononuclear cell isolation from lungs (D) followed by flow cytometric phenotypic characterization of the mononuclear cell population (E). The data depicted is the mean ± S.D. of the lung mononuclear cell number from a representative experiment. Asterisks indicate statistically significant difference when compared to the PBS-exposed controls, # indicates statistically significant difference when compared to SEB-exposed isotype mAb-treated CD44WT mice p ≤ 0.05.

induced model of airway disease through alteration of macrophage recruitment to the lungs [11]. Our current study supports these findings by demonstrating that deletion of CD44 leads to significant protection from SEB-induced acute lung inflammation. Similar to other studies, this was characterized by significantly reduced accumulation of inflammatory cells, including T cells, NK cells, NKT cells,

macrophage, and neutrophils. In addition, deletion of CD44 led to a significant attenuation of SEB-induced cytokine production. The reduced production of cytokines seen in the lungs of SEB-exposed CD44KO mice was not specific for Th1, Th2, or Th17 cytokines as all cytokines induced by SEB were reduced. In addition, we demonstrated that the effects seen in CD44KO mice could be, to some extent, reproduced using

50

Figure 8 Treatment with anti-CD44 mAbs leads to a reduction in SEB-induced inflammatory cytokine levels in the lungs. The effect of anti-CD44 mAbs on SEB-induced vascular permeability in vivo was determined by exposing mice to SEB, as described in the Materials and methods section, and treating the mice with anti-CD44 mAbs (50 μg/mouse i.p.) or isotype control mAbs (50 μg/mouse i.p.) immediately following SEB exposure. The levels of BALF cytokine protein levels and total lung cytokine mRNA were determined 24 h later. The protein levels of cytokines in BALF were determined using a cytokine bead array (A). Cytokine mRNA levels in whole lung extracts were determined by real-time RT-PCR (B). Asterisks indicate statistically significant difference when compared to the cytokine levels from SEB-exposed isotype control mAbs treated CD44WT mice, p ≤ 0.05.

anti-CD44 mAbs, suggesting that it might be possible to use CD44-specific antibody treatment for lung inflammation associated with SEB exposure. In contrast to these findings, CD44 has been reported to potentially act in a protective manner in lung inflammation. For example, intratracheal exposure to LPS led to an increased inflammatory response in CD44KO mice [25]. The reasons for the differences in the reported role of CD44 in this LPS-induced lung inflammation model remain unclear but may be due to differences in stimuli (SEB vs. LPS), dose (high vs. low LPS) and/or route of administration.

J. Sun et al. The influence of CD44 on SEB-induced lung inflammation could be due to a number of possibilities, including defective SEB-induced activation, reduced migration of inflammatory cells to the lungs, reduced cytokine production, reduced adhesion, and/or elevated levels of apoptosis in CD44KO stimulated leukocytes. Although many of these mechanisms are not independent of one another, we were able to provide evidence to suggest that deletion of CD44 did not have a direct effect on SEB-induced proliferation or apoptosis. Furthermore, we provide evidence to suggest that the reduced inflammatory response seen in CD44KO mice was due, at least in part, to reduced cytokine production and a reduced binding ability of SEB-activated leukocytes. Whether the increased inflammatory response was a direct result of CD44 binding to ligands on epithelial cells or indirectly through increased expression of other adhesion molecules regulated directly by CD44 signaling or by cytokines produced in response to CD44 signaling remains to be elucidated. All of these possibilities are supported by previous studies examining the role of CD44 in the inflammatory response. A number of studies have demonstrated a direct role of CD44 as an adhesion molecule important for migration of various cells including immune cells [42]. In addition, reports demonstrate that CD44 can influence the expression of other adhesion molecules. For example, signaling through CD44 has been shown to increase the expression of ICAM-1 and VCAM-1 [43,44]. Furthermore, signaling through CD44 has been reported to play an important role in the production of inflammatory cytokines, such as IL-6, which may directly influence the expression of other adhesion molecules important for leukocyte migration to the lungs, while other reports suggests an important role of CD44 in the production of IFN-γ [18,20,45,46]. Hyaluronic acid (HA), the principal ligand for CD44, may play an important role in the development of lung inflammation. An interesting emerging concept suggests the possibility that LWM-HA has pro-inflammatory activity while HWM-HA has anti-inflammatory properties. For example, the production of low molecular weight HA (LMW-HA) was reported to be elevated in airway fibroblast from asthma patients [47]. Furthermore, it has been shown that treatment with LWM-HA leads to increased LPS-induced lung inflammation either directly or following priming with ozone [48]. However, the importance of LMW-HA in SEB-induced lung inflammation remains unclear. We speculate that SEBexposure may lead to increased ROS leading to depolymerization of HMW-HA into LMW-HA and subsequent signaling through CD44. This possibility is supported by the observation that exposure to SEB leads to increased ROS and by separate studies demonstrating that ROS can lead to elevated levels of LMW-HA [49–51]. In the current study, we demonstrated that exposure of CD44WT mice to SEB led to increased expression of CD44 in lung mononuclear cells. In previous work from our laboratory we reported that exposure of CD44WT mice to high dose IL-2 led to a similar increase in CD44 expression. Furthermore, we demonstrated that the increase in CD44 was, at least in part, due to the increased expression of CD44 isoforms. CD44 exists as a number of isoforms, which differ in their molecular weights, ranging from 85 to 260 kDa [5,6]. The most common form has a molecular weight of 85–90 kDa and is referred to as wild-type, standard (CD44s), or the

CD44 for the treatment of SEBnduced ALI hematopoietic form [52]. Naïve leukocytes primarily express CD44s. However, recent evidence has shown the existence of a number of isoforms which are formed due to alternative splicing among variant exons. Therefore, it is possible that the our reported increase of CD44 expression in lung mononuclear cells following SEB exposure, in part, may be due to increased expression of specific CD44 isoforms. Furthermore, we report a significant increase in the CD44 high expressing cells in the lungs following exposure to SEB. A similar increase in CD44 high cells was reported in a study examining the role of CD44 in lymphokine-activated killer cell lysis of melanoma cells, which was directly correlated with an increase in CD44 isoform expression and elevated effector cell activity [38]. Although the contribution of various CD44 isoforms to SEB-induced lung injury remains unclear, the similar increase in CD44 high expressing cells seen in the current study suggests the possibility that CD44 isoforms may play an important role in SEB-induced lung injury. Interestingly, the ability of CD44 to bind other ligands is influenced by the expression of CD44 isoforms. For example, the CD44v6 isoform is capable of binding to osteopontin, which has been reported to have proinflammatory activity in the lungs. Therefore, further exploring the role of CD44 isoforms in SEB-induce lung injury may lead to a better understanding of lung inflammation and may ultimately lead to novel targets for treating inflammatory conditions in the lungs. In summary, acute inflammation and the resulting lung injury are major complications that result from exposure to staphylococcal enterotoxins such as SEB. To date, there are no effective treatments for the resulting inflammatory response. In the current study we report on the potential importance of CD44 in the development of SEB-associated lung injury. Furthermore, results from our study suggest that targeting CD44 may be an effective strategy for treating and/or preventing SEB-induced lung injury.

Conflict of interest statement The authors declare that there are no conflicts of interest.

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