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Keratinocyte growth factor-2 inhibits bacterial infection with Pseudomonas aeruginosa pneumonia in a mouse model
J Infect Chemother 22 (2016) 44e52
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Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
Keratinocyte growth factor-2 inhibits bacterial infection with Pseudomonas aeruginosa pneumonia in a mouse model Nana Feng a, Qin Wang a, Jian Zhou a, Jing Li a, Xiaoxing Wen a, Shujing Chen a, Zhenhua Zhu a, Chunxue Bai a, Yuanlin Song a, b, *, Huayin Li a, ** a b
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China Department of Pulmonary Medicine, Zhongshan Hospital, Qingpu Branch, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 May 2015 Received in revised form 24 September 2015 Accepted 14 October 2015 Available online 24 November 2015
To determine protective effects of concurrent administration of Keratinocyte growth factor-2 (KGF-2) with Pseudomonas aeruginosa (P. aeruginosa) inoculation on the induced pneumonia. KGF-2 (5 mg/kg) was concurrently administered into the left lobe of 55 mice with P. aeruginosa PAO1 (5 106 CFU, half-lethal dose); 55 mice in the control group were concurrently administered PBS with the PAO1. We detected and analyzed: body temperature; amount of P. aeruginosa in homogenates; count of total number of nucleated cells and of mononuclear macrophages; protein concentration in bronchoalveolar lavage fluid (BALF); lung wet-to-dry weight ratio; cytokines in BALF and blood; and lung morphology. To study survival rate, concurrent administration of KGF-2 (experimental group) versus PBS (control) with a lethal dose of PAO1 (1 107 CFU was performed, and survivorship was documented for 7 days post-inoculation. The bacterial CFU in lung homogenates was significantly decreased in the KGF-2 group compared to the control group. There were significantly more mononuclear macrophages in the BALF from the KGF-2 group than from the control group (p < 0.05). KGF-2 increased the surfactant protein and GM-CSF mRNA in lung at 6 h and 72 h after inoculation. Significant reduction of lung injury scores, protein concentrations, lung wet-to-dry weight ratio, and IL-6 and TNF-a levels was noted in the KGF-2 treated rats at 72 h after inoculation (p < 0.05). The 7-day survival rate of the KGF-2 group was significantly higher than that of the control group (p < 0.05). Concurrent administration of KGF-2 facilitates the clearance of P. aeruginosa from the lungs, attenuates P. aeruginosa-induced lung injury, and extends the 7-day survival rate in mice model with P. aeruginosa pneumonia. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Keratinocyte growth factor-2 Pseudomonas aeruginosa Pneumonia Lung injury
1. Introduction Pseudomonas aeruginosa (P. aeruginosa), an opportunistic pathogen, frequently causes lower airway infection in patients with impaired immunity [1]. Antibiotic-resistant strains of P. aeruginosa are emerging and spreading in many countries. Infection with
* Corresponding author. Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Tel.: þ86 21 64041990; fax: þ86 21 64187165. ** Corresponding author. Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Tel.: þ86 21 64041990; fax: þ86 21 64187165. E-mail addresses: [email protected] (Y. Song), [email protected] (H. Li).
multidrug-resistant (MDR) and extensively drug-resistant (EDR) isolates that are insensitive to all standard antipseudomonal antibiotics (including carbapenems, fluoroquinolone and aminoglycosides) except colistin represents a special challenge, as physicians may have exhausted all effective antibiotics in treating P. aeruginosa infection [2,3]. New therapeutics, that can potently inhibit P. aeruginosa infection but do not frequently generate drug resistance, are urgently needed. Keratinocyte growth factor-2 (KGF-2), a member of the fibroblast growth factor family, is a 208 amino acid polypeptide, also known as fibroblast growth factor-10 (FGF-10) [4]. Similar to KGF, KGF-2 is mainly expressed and secreted by stromal cells; once released, it binds with high affinity to FGF receptors 2IIIb (FGFR2III-b) and 1III-b (FGFR1III-b) that are expressed exclusively on epithelial and endothelial cells [5]. KGF-2 is also similar to KGF in
http://dx.doi.org/10.1016/j.jiac.2015.10.005 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
N. Feng et al. / J Infect Chemother 22 (2016) 44e52
structure and biological function. Thus, KGF-2 acts as a paracrine mediator, facilitating epithelial cell proliferation/differentiation, angiogenesis and barrier function of capillary monolayers [6e8]. KGF-2 was found to accelerate healing of corneal epithelial wounds [9] and incisional wounds in vivo [10] and to reduce inflammatory bowel disease in a preclinical model [11]. These known biological functions of KGF-2 deem it a potential therapeutic. Recently, KGF-2 has been in phase II trials for topical treatment of chronic venous ulcers and systemic treatment of ulcerative colitis [12]. KGF-2 also prevented mucositis in patients who received chemotherapy after bone marrow transplantation [13]. Prophylactic application of KGF-2 requires further investigation. Our recent studies demonstrated that pretreatment with KGF-2 attenuated severe pulmonary edema [14] and lung injuries induced by ischemia/reperfusion [15], ventilator [16] and lipopolysaccharide [17] in rats. Viget et al. [18] showed that KGF pretreatment had antibacterial effects at the acute stage of P. aeruginosa pneumonia in rats. Wu et al. [19] also found KGF pretreatment could facilitate clearing Gram-negative pathogens from the lung within 24 h post-infection. Lee et al. [20] showed that KGF secreted by mesenchymal stem cells could kill E. coli perfused from human lungs under ex vivo culture conditions. Those results suggest that KGF-2 had great potential as a prophylactic and therapeutic, but definite effects of KGF-2 in inhibiting P. aeruginosa infection in the lungs remains to be established. The aim of this study was to determine whether concurrent administration of KGF2 with P. aeruginosa could inhibit bacterial infection and mitigate P. aeruginosa pneumonia in early (6-h) and late (24-h and 72-h) pneumonia after pathogen instillation in a mouse model. 2. Materials and methods 2.1. Animals A total of 180 female BALB/c mice, 6e8 weeks old and certified as specific pathogen-free (SLAC Laboratory Animal, China), were housed in Zhongshan Hospital Animal Care Facility. All animals had access to food and water ad libitum. The experimental protocol was approved by the Animal Care and Use Committee, Fudan University, Shanghai. 2.2. Bacteria This study used the P. aeruginosa PAO1 strain, for which the lethal dose was previously titrated 1 107 CFU (unpublished data). P. aeruginosa was incubated in tryptic soy broth (Oxide Microbiology Products, England) at 37 C; then the culture was washed twice and resuspended in sterile PBS. 2.3. Experiment grouping 2.3.1. Half-lethal dose of P. aeruginosa and KGF-2 One hundred and ten mice were randomly divided into an experimental group (intratracheal administration of P. aeruginosa and KGF-2) and a control group (intratracheal administration of P. aeruginosa and PBS). In the experimental group, recombinant human KGF-2 (rhKGF-2, 5 mg/kg in 0.05 ml; New Summit Biopharma, Shanghai, China) was administered to mice concurrently with inoculation with P. aeruginosa (5 106 CFU, half-lethal dose) by the intratracheal route. 2.3.2. Lethal dose of P. aeruginosa and KGF-2 To study the effect of KGF-2 on survival of P. aeruginosa-infected mice, the remaining 40 mice were randomly divided into a experimental group (intratracheal instillation of 1 107 CFU P. aeruginosa
45
and rhKGF-2, 5 mg/kg in 0.05 ml; n ¼ 20) and a control group (intratracheal instillation of 1 107 CFU P. aeruginosa and 0.05 ml PBS; n ¼ 20). The survival of mice in all groups was documented for 7 days after inoculation. 2.3.3. KGF-2 and PBS To study the effect of KGF-2 on the total cell number and the macrophages counts in BALF, 10 mice were randomly divided into a KGF-2þPBS group (rhKGF-2, 5 mg/kg in 0.05 ml) and PBS group (PBS,0.05 ml). 2.4. Intratracheal instillation Mice were inoculated by instilling the bacteria with KGF-2 or PBS into the left lung as previously described [21]. Briefly, following anesthesia with avertin, mice were placed on a board with their heads elevated at 45 . Then 50 ml of bacterial working solution was instilled into the left lung through the trachea using a 22G gavage needle. Mice were recovered for 15 min prior to replacement into their cages. All animals were active and appeared normal in 30 min post-inoculation. 2.5. Monitor of rectal temperature Rectal temperature was hourly monitored for initial 12 h post bacterial instillation, then daily for 7 days (5 mice for each group). 2.6. Lung and blood bacterial enumeration At 6, 24, and 72 h after inoculation, 5 mice per group in the halflethal dose experiment were euthanized with a high dosage of avertin. Blood and lungs were collected under sterile conditions. The left lung was removed and placed in 1 ml of sterile PBS and homogenized for quantification of bacteria. Serial ten-fold dilutions of lung homogenates and whole blood in sterile PBS were carried out. Then different dilutions were spread onto pseudomonas centrimide agar plates (Oxide Microbiology Products, England) for overnight incubation at 37 C prior to enumeration. 2.7. Lung wet to dry weight ratio (W/D) Mice were euthanized with averting, the left lung was isolated and the wet weight was recorded. The lungs were then placed in a 60 C incubator for 3 days, during which the dry weight was recorded to determine the wet-to-dry weight ratio. 2.8. Bronchoalveolar lavage fluid Upon euthanasia of 5 mice per group in the half-lethal dose experiment at each time point described above, bronchoalveolar lavage fluid (BALF) was also collected prior to lung removal from the left lung by cannulating the trachea and gently flushing the left lung three times with 0.5 ml PBS. BALF was immediately centrifuged at 1500 g for 10 min at 4 C, then the supernatant was stored at 80 C for further analysis, while the cell pellet was used for cell cytospin preparation. 2.9. Total cell count and differential cell count The total number of nucleated cells in BALF was counted with hemocytometer. The resuspended BALF cells were centrifuged, transferred to slides and stained with Wright'seGiemsa (Jiancheng, Nanjing, China). Finally, leukocytes and macrophages on all slides were quantified by counting a total of 200 cells/slide at 40 magnification.
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2.10. Protein concentration and cytokine levels in BALF Total amount of proteins in BALF was measured using the bicinchoninic acid protein assay kit (Thermo Scientific, Pittsburgh, PA, USA). Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) concentrations in BALF and plasma were measured by an ELISA kit (R&D Systems, USA). All procedures followed the instructions provided by each kit. 2.11. Lung histopathology In the half-lethal dose experiment, another 5 mice per group were euthanized at 6, 24, and 72 h after inoculation with a high dose of avertin. The left lung was surgically removed and fixed in 10% formalin for histology. Sections were stained with Hematoxylin and eosin (H&E). The lung injury was scored by a pathologist who was blind to sample ID [22]. 2.12. Quantitative real-time polymerase chain reaction analyses Total RNA was extracted using the Trizol (Invitrogen, CA, USA). RNA was reverse-transcribed, and real-time polymerase chain reaction (qPCR) was performed using primers for the examined transcripts (Table 1). 2.13. Statistical analysis Quantitative data was presented as mean ± SD and analyzed using SPSS v18.0 (New York, IBM, USA). Comparisons among multiple groups were performed with one-way ANOVAs, while comparisons between two groups used the ManneWhitney U test. Survival rates were plotted on KaplaneMeier product limit curves and compared by log-rank test. A p value 0.05 was considered statistically significant. 3. Results 3.1. KGF-2 alleviated lung injury in the Pseudomonas aeruginosa pneumonia Lung edema, inflammatory cell infiltration, alveolar hemorrhage and hyaline membrane formation were evident in the alveolar space at 6 h (Fig. 1A), 24 h (Fig. 1B) and 72 h (Fig. 1C) after bacterial infection in both control and KGF-2 groups. However, average lung injury score in the KGF-2 group was significantly lower than the control group at 72 h after inoculation (p < 0.05, Fig. 1D). 3.2. KGF-2 facilitated bacterial clearance from the infected lung Average body temperature in mice with PAO1-induced pneumonia was decreased in the first 6 h post-inoculation for both the
control and experimental groups; however, the body temperature of mice in the study group was significantly higher than those in the control group (p < 0.01, Fig. 2A), suggesting KGF-2 administration attenuated the decline in body temperature within 24 h of the pneumonia. Mice body temperature from both groups returned to normal over a 3-day period. Furthermore, the total number of CFUs detected in the lungs was significantly lower in the KGF-2 group at 6, 24 and 72 h after inoculation compared to the control group (p < 0.05, Fig. 2B). However, there was no viable bacteria in blood samples and the mice did not develop bacteremia.
3.3. Effect of KGF-2 on lung injury indices following intratracheal P. aeruginosa We also determined the lung injury through analyzing lung wet/ dry weight ratios. At 6 h, 12 h and 72 h post-infection, the lung wetto-dry weight ratios of left lungs from animals with untreated control group were significantly higher than those treated with KGF-2 (Fig. 3A, p < 0.05). There was evidence of significantly reduced injury (p < 0.05) in the KGF-2 group comparing the control group in all analyses including the concentration of total proteins (Fig. 3B, p < 0.05). We analyzed the number of cells in BALF. No significant difference in the total number of nucleated cells in BALF was noted between two groups (Fig. 3C). However, the number of macrophages in the KGF-2 group was significantly higher compared to the control group (p < 0.05, Fig. 3D) at 6, 24 and 72 h after inoculation.
3.4. KGF-2 reduces inflammatory process in P. aeruginosa-induced lung injury The number of neutrophils in the KGF-2 group was significantly lower at 24 and 72 h after inoculation than in the control group (p < 0.05, Fig. 4A). There was evidence of significantly reduced injury (p < 0.05) in the KGF-2 group comparing the control group in all analyses including the concentration of IL-6 in BALF at 24 and 72 h (Fig. 4B), IL-6 in plasma at 24 and 72 h (Fig. 4C), TNF-a in BALF at 24 h (Fig. 4D), and TNF-a in plasma at 24 and 72 h (Fig. 4E). 3.5. KGF-2 restores SP-A, SP-C and GM-CSF synthesis after P. aeruginosa challenge KGF-2 treatment significantly increased expression levels of SPA, surfactant protein C (SP-C) and GM-CSF in the lung compared with control group at 6 h and 72 h after inoculation (Fig. 5). KGF-2 treatment significantly increased SP-A, SP-C mRNA and GM-CSF mRNA expression compared with control group at 6 h and 72 h after inoculation (Fig. 5A, B and C).
Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Genes
Primers (50 -30 )
GenBank accession no.
Amplicon size (bp)
Surfactant protein A
F-CATAAGGAAGCCCAGGAACA R-TCAGCAAACCCTCAACAGC F-CTGATGGAGAGTCCACCGGA R-ATC ACCACGACAACGAGGAC F-CCCTGGAGTTCTGTGGTCA R-GGGTGCTGAGAGGCTGTAGA F-AGGTCGGTGTGAACGGATTTG R-GGGGTCGTTGATGGCAACA
NM_023134.4
127
NM_011359.2
125
NM_009969.4
164
NM_008084.2
92
Surfactant protein C GM-CSF GAPDH
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); granulocyte-macrophage coloy-stimulating factor (GM-CSF).
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Fig. 1. Histological changes in control and KGF-2 group (magnification 200). Hematoxylin and eosin (H&E) staining of the lung tissue in control and KGF-2 group 6 h (A), 24 h (B), and 72 h (C) after inoculation. (D) Mean lung score for each group at 6 h, 24 h and 72 h after inoculation (n ¼ 5 mice per group). The data are presented as mean ± SD. *p < 0.05.
3.6. The number of total cells and differential cells in BALF following intratracheal administration of KGF-2 Mice were treated with KGF-2 alone or PBS, and then sacrificed and lavaged, the number of cells was analyzed in BALF. The number of total cells in KGF-2þPBS group was significantly higher compared to PBS group at 6, 24 and 72 h after inoculation (p < 0.01,
Fig. 6A). The percentage of macrophages in PBS group were significantly higher than those in KGF-2þPBS group at 6 h and 72 h after inoculation (p < 0.01, Fig. 6B). The number of macrophages in the KGF-2þPBS group was significantly higher compared to the control group at 6, 24 and 72 h after inoculation (p < 0.01, Fig. 6C). The percentage of neutrophils in KGF-2þPBS group were significantly higher than those in PBS group at 6 h and 24 h after
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higher compared to PBS group at 6 and 24 after inoculation (p < 0.01, Fig. 6E). However, the number of neutrophils in PBS group were significantly higher than those in KGF-2þPBS group at 72 h after inoculation (p < 0.01, Fig. 6E). 3.7. KGF-2 improved mouse survival rate For the lethal-dose experiment, the survival rate in the KGF-2 group was significantly higher at 75% than 50% in the control group (p < 0.05, Fig. 7). 4. Discussion
Fig. 2. (A) Body temperature curves after PAO1 infection. The rectal temperature were measured and plotted at 6 h, 24 h and 72 h (n ¼ 5 mice per group). (B) The total P. aeruginosa CFUs in the lungs at 6 h, 24 h, 72 h after inoculation (n ¼ 5 mice per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01.
inoculation (p < 0.01, Fig. 6D). However, the percentage of neutrophils in PBS group were significantly higher than those in KGF2þPBS group at 72 h after inoculation (p < 0.01, Fig. 6D). The number of neutrophils in KGF-2þPBS group was significantly
The major purpose of this study was to determine if KGF-2 administration concurrent with bacterial inoculation can mitigate P. aeruginosa infection and its pathological injury in the lung of a mouse model. We found that pulmonary injury associated with mild to moderate pneumonia was attenuated at the late stage in the KGF-2 treated mice compared to the control mice. The reduction of lung injury with KGF-2 treatment was supported by significantly reduced pulmonary edema and other histological changes. KGF-2 treatment also attenuated the circulation leakage from the P. aeruginosa-infected tissues including the total BALF protein and lung wet-to-dry weight ratio, suggesting the protective effects of KGF-2 on lung epithelial barrier. Although previous study showed that KGF pretreatment can protect rats from P. aeruginosa-induced pneumonia [22]. A major concern about this treatment approach was the different kinetics between bacterial infection and the protective effects of KGF-2. The present work confirms that lung injury and inflammation were induced 6 h after the inoculation with P. aeruginosa in mice. Previous study also showed that a typical pneumonia occurs 6 h after P. aeruginosa was instilled into the lung [23]. Additionally, there were technical concerns. In this study, the protective effects of KGF-2 did not become detectable until 72 h after inoculation, which was consistent with the previous suggestion that it may take 3 days for KGF-2 to exhibit strong therapeutic effects while a minimum duration of two days is needed for KGF-2
Fig. 3. KGF-2 improves PAO1-induced protein-rich edema. (A) Lung W/D weight ratios in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. W/D ratio in control group was significantly higher than in KGF-2 group at 72 h after inoculation (B) The total protein concentration recovered in BALF. Total protein contents in control group were significantly higher than those in KGF-2 group at 6 h, 24 h and 72 h after inoculation. (C) Total white blood cell counts in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. (D) The number of macrophages in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. The number of macrophages in KGF-2 group were significantly higher than those in control group at 6 h, 24 h and 72 h after inoculation (n ¼ 5 mice per group). The data are presented as mean ± SD. *p < 0.05.
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Fig. 4. KGF-2 reduces neutrophils infiltration and inflammatory cytokines in the BALF. (A) The number of neutrophils in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. Concentrations of cytokines in BALF and plasma of the KGF-2 and control groups at 6 h, 24 h, and 72 h after inoculation (n ¼ 5 mice per group). (B) The concentration of IL-6 in BALF. (C) The concentration of IL-6 in plasma culation. (D) The concentration of TNF-a in BALF. (E) The concentration of TNF-a in plasma (n ¼ 5 mice per group). The data are shown as mean ± SD. *p < 0.05 **p < 0.01.
Fig. 5. SP-A, SP-C and GM-CSF mRNA level at 6 h and 72 h after inoculation. (A) SP-A mRNA expression in the lung. (B) SP-C mRNA expression in the lung. (C) GM-CSF mRNA expression in the lung. *p < 0.05 **p < 0.01.
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Fig. 6. The number of total cells and differential cells in BALF following intratracheal KGF-2 administration. (A) Total white blood cell counts in KGF-2þPBS group and PBS group at 6 h, 24 h and 72 h after inoculation (P < 0.01). (B) The percentage of macrophages in KGF-2þPBS and PBS group at 6 h, 24 h and 72 h after inoculation. (C) The number of macrophages in KGF-2þPBS and PBS group at 6 h, 24 h and 72 h after inoculation. (D) The percentage of neutrophils in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. (E)The number of neutrophils in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation (n ¼ 5 mice per group). The data are presented as mean ± SD. *p < 0.05 **p < 0.01 ***p < 0.001.
to reach full effect [14e17]. The mechanisms underlying the protective effects of KGF-2 in P. aeruginosa-induced acute lung injury remain incompletely understood. The present study investigated the role of KGF-2 in reducing the bacteria number and lung inflammation caused by P. aeruginosa instillation, and changes in producing surfactant proteins. We also noted that inclusion of KGF-2 in the inoculum increased bacterial clearance in the lung as observed in the early and late
Fig. 7. Survival rates of mice in control and KGF-2 groups. The 7-day survival rate was calculated and plotted following PAO1 (1 107 CFU)-induced pneumonia in the control and KGF-2 groups (n ¼ 20 mice per group). *p < 0.05.
phase of P. aeruginosa pneumonia, suggesting that local administration of KGF-2 may potentially function as an adjunct therapy to antibiotics for mild pneumonic patients infected with multidrugresistant bacteria. There are likely multiple mechanisms underlying the effect of inhibiting bacteria, though exactly what they may be remains unclear. To study the mechanisms underlying the favorable effects of KGF-2 in reducing bacterial number, we investigate the effect of KGF-2 on the total cell number, the macrophages counts and the number of neutrophils in BALF. In our study, the number of macrophages and neutrophils in the KGF-2þPBS group was significantly higher compared to the PBS group at 6, 24 and 72 h after inoculation. Ferreira et al. [24] showed that bacterial viability was reduced in the presence of KGF; however, KGF alone did not show any inhibiting effect on P. aeruginosa. Our data suggests that KGF-2 increase the number of macrophages at 6, 24 and 72 h after inoculation. Studies [16,17] examined the expression of surfactant proteins (SP) in lung tissue and found that KGF-2 pretreatment partially restored SP-A and SP-C expression. Thus we examined the SP-A and SP-C mRNA level in lung tissue and demonstrated that KGF-2 treatment partially restored SP-A and SP-C mRNA expression at early and late phases of pneumonia. The increased mRNA expression of surfactant protein might be attributed to alveolar type II
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epithelial cell proliferation and differentiation, and might indirectly prevent a permeability defect which could be a potential mechanism to deliver KGF-2 therapeutic benefits. Another study [25] showed that surfactant protein A acted as an opsonin to increase the number of macrophages in BALF and stimulate macrophage phagocytosis of a live mucoid strain of P. aeruginosa at early and late phases of pneumonia. A mechanistic investigation [19] demonstrated that KGF augmented the host defenses to E. coli and P. aeruginosa in the lungs, partly through GM-CSF-dependent macrophage recruitment and activation. Thus we also examined the GM-CSF mRNA level in lung tissue and demonstrated that KGF-2 treatment partially restored SP-A and SP-C mRNA expression. A report by Lee et al. [20] also showed that KGF increased bacterial killing through increased alveolar macrophage phagocytosis and decreased apoptosis of human monocytes by AKT phosphorylation. The inflammatory response was attenuated in late P. aeruginosa pneumonia of mice treated with KGF-2. The neutrophil counts in BALF collected in the late phase of pneumonia were significantly lower in the KGF-2 group than in the control group. Several pro-inflammation cytokines, including IL-6, TNF-a and IL1b, are involved in the initiation and development of pulmonary inflammatory response. We found that the TNF-a and IL-6 levels in BALF and blood were decreased in the KGF-2 group. KGF pretreatment reduced the blood neutrophil counts at day 3 post-injury [26] and pulmonary neutrophil counts within 8 h post-injury [27]. We previously demonstrated that a protective effect of KGF-2 pretreatment in a VILI rat model was probably mediated through decreased release of inflammatory cytokines, accompanied by reduced neutrophil infiltration and activation [16]. Thus, KGF-2 may attenuate pneumonia-associated lung inflammation by suppressing the expression and release of proinflammatory cytokines, reduces the signals attracting neutrophils to the lungs. Finally, our data suggests that the concurrent administration of KGF-2 improved survival rate of mice with lethal pneumonia. This observation was in agreement with the report that the survival was significantly increased in severe pneumonic rats with KGF pretreatment [18]. Multi-drug resistant P. aeruginosa infection is a severe problem in clinical practices associated with high mortality and morbidity [28], and the current effective treatment is very limited, thus a new therapeutic approach with promising outcomes is highly demand. One limitation of this study was that we only investigated the protective effects of concurrent administration of KGF-2 for kinetic and technical reasons. The concurrent administration of both KGF-2 and inoculum reduces the number of animal deaths because the mice easily died of repeated intratracheal intubation if KGF-2 was administered after the inoculation. A fine mixture of PAO1 and KGF2 made before the instillation can be evenly distributed to the same sites of the lung, an important requirement for minimizing procedural variation and achieving maximal effect. Our reasoning was that it would favor further testing of the therapeutic effects of KGF2 in the rat model if this study further confirmed the protection against P. aeruginosa infection. We are encouraged by our results and, in the future, will test the therapeutic effects of KGF-2 administrated after infection. In conclusion, in this study, we found that concurrent administration of KGF-2 exhibited protective effects through increased bacterial clearance and reduced lung injury in mild to moderate pneumonia. KGF-2 also increased the survival rate in mice with severe P. aeruginosa pneumonia. Our results suggest the local administration of KGF-2 may function as an adjunct therapy to antibiotics in patients with P. aeruginosa pneumonia.
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Conflict of interest The authors report no conflicts of interest. The authors are solely responsible for the contents and interpretation of the results. Acknowledgments The authors thank the members of the Bai laboratory for their invaluable advice and discussions. The KGF-2 was a gift from Xinshengyuan Pharmaceutical Company, China. The work was partly supported by Shanghai Leading Academic Discipline Project (Project Number: B115), The National Nature Science Foundation of China (811700056, 81400018, 81400002), The Shanghai Committee of Science and Technology (11411951102, 12JC1402300), and the Academic Special Science and Research Foundation for PhD Education (20130071110044). References [1] Coggan KA, Wolfgang MC. Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype. Curr Issues Mol Biol 2012;14:47e70. [2] Cabot G, Ocampo-Sosa AA, Dominguez MA, Gago JF, Juan C, Tubau F, et al. Genetic markers of widespread extensively drug-resistant Pseudomonas aeruginosa high-risk clones. Antimicrob Agents Chemother 2012;56: 6349e57. [3] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268e81. [4] Yamasaki M, Miyake A, Tagashira S, Itoh N. Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family. J Biol Chem 1996;271:15918e21. [5] Emoto H, Tagashira S, Mattei MG, Yamasaki M, Hashimoto G, Katsumata T, et al. Structure and expression of human fibroblast growth factor-10. J Biol Chem 1997;272:23191e4. [6] Ishiwata T, Naito Z, Lu YP, Kawahara K, Fujii T, Kawamoto Y, et al. Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginineinduced acute pancreatitis. Exp Mol Pathol 2002;73:181e90. [7] Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, et al. Fgf10 is essential for limb and lung formation. Nat Genet 1999;21:138e41. [8] Igarashi M, Finch PW, Aaronson SA. Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J Biol Chem 1998;273:13230e5. [9] Wang X, Zhou X, Ma J, Tian H, Jiao Y, Zhang R, et al. Effects of keratinocyte growth factor-2 on corneal epithelial wound healing in a rabbit model of carbon dioxide laser injury. Biol Pharm Bull 2010;33:971e6. [10] Jimenez PA, Rampy MA. Keratinocyte growth factor-2 accelerates wound healing in incisional wounds. J Surg Res 1999;81:238e42. [11] Miceli R, Hubert M, Santiago G, Yao DL, Coleman TA, Huddleston KA, et al. Efficacy of keratinocyte growth factor-2 in dextran sulfate sodium-induced murine colitis. J Pharmacol Exp Ther 1999;290:464e71. [12] Robson MC, Phillips TJ, Falanga V, Odenheimer DJ, Parish LC, Jensen JL, et al. Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Repair Regen 2001;9:347e52. [13] Freytes CO, Ratanatharathorn V, Taylor C, Abboud C, Chesser N, Restrepo A, et al. Phase I/II randomized trial evaluating the safety and clinical effects of repifermin administered to reduce mucositis in patients undergoing autologous hematopoietic stem cell transplantation. Clin Cancer Res 2004;10: 8318e24. [14] She J, Goolaerts A, Shen J, Bi J, Tong L, Gao L, et al. KGF-2 targets alveolar epithelia and capillary endothelia to reduce high altitude pulmonary oedema in rats. J Cell Mol Med 2012;16:3074e84. [15] Fang X, Wang L, Shi L, Chen C, Wang Q, Bai C, et al. Protective Effects of KGF-2 on Ischemia/Reperfusion-Induced Lung Injury in Rats. Am J Respir Cell Mol Biol 2014;6:1156e65. [16] Bi J, Tong L, Zhu X, Yang D, Bai C, Song Y, et al. Keratinocyte growth factor-2 intratracheal instillation significantly attenuates ventilator-induced lung injury in rats. J Cell Mol Med 2014;18:1226e35. [17] Tong L, Bi J, Zhu X, Wang G, Liu J, Rong L, et al. Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats. Respir Physiol Neurobiol 2014;201:7e14. [18] Viget NB, Guery BP, Ader F, Neviere R, Alfandari S, Creuzy C, et al. Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2000;279:L1199e209. [19] Wu H, Suzuki T, Carey B, Trapnell BC, McCormack FX. Keratinocyte growth factor augments pulmonary innate immunity through epithelium-driven,
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N. Feng et al. / J Infect Chemother 22 (2016) 44e52 GM-CSF-dependent paracrine activation of alveolar macrophages. J Biol Chem 2011;286:14932e40. Lee JW, Krasnodembskaya A, McKenna DH, Song Y, Abbott J, Matthay MA. Therapeutic effects of human mesenchymal stem cells in ex vivo human lungs injured with live bacteria. Am J Respir Crit Care Med 2013;187: 751e60. Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, Wiener-Kronish JP, et al. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat Med 1999;5: 392e8. Welsh DA, Summer WR, Dobard EP, Nelson S, Mason CM. Keratinocyte growth factor prevents ventilator-induced lung injury in an ex vivo rat model. Am J Respir Crit Care Med 2000;162:1081e6. Song Y, Baer M, Srinivasan R, Lima J, Yarranton G, Bebbington C, et al. PcrV antibody-antibiotic combination improves survival in Pseudomonas aeruginosa-infected mice. Eur J Clin Microbiol Infect Dis 2012;31:1837e45.
[24] Sobral CS, Gragnani A, Morgan J, Ferreira LM. Inhibition of proliferation of Pseudomonas aeruginosa by KGF in an experimental burn model using human cultured keratinocytes. Burns 2007;33:613e20. [25] Mariencheck WI, Savov J, Dong Q, Tino MJ, Wright JR. Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain of P. aeruginosa. Am J Physiol 1999;277:L777e86. [26] Yano T, Deterding RR, Simonet WS, Shannon JM, Mason RJ. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am J Respir Cell Mol Biol 1996;15:433e42. [27] Nemzek JA, Ebong SJ, Kim J, Bolgos GL, Remick DG. Keratinocyte growth factor pretreatment is associated with decreased macrophage inflammatory protein-2alpha concentrations and reduced neutrophil recruitment in acid aspiration lung injury. Shock 2002;18:501e6. [28] Chung DR, Song JH, Kim SH, Thamlikitkul V, Huang SG, Wang H, et al. High prevalence of multidrug-resistant nonfermenters in hospital-acquired pneumonia in Asia. Am J Respir Crit Care Med 2011;184:1409e17.
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
Keratinocyte growth factor-2 inhibits bacterial infection with Pseudomonas aeruginosa pneumonia in a mouse model Nana Feng a, Qin Wang a, Jian Zhou a, Jing Li a, Xiaoxing Wen a, Shujing Chen a, Zhenhua Zhu a, Chunxue Bai a, Yuanlin Song a, b, *, Huayin Li a, ** a b
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China Department of Pulmonary Medicine, Zhongshan Hospital, Qingpu Branch, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 May 2015 Received in revised form 24 September 2015 Accepted 14 October 2015 Available online 24 November 2015
To determine protective effects of concurrent administration of Keratinocyte growth factor-2 (KGF-2) with Pseudomonas aeruginosa (P. aeruginosa) inoculation on the induced pneumonia. KGF-2 (5 mg/kg) was concurrently administered into the left lobe of 55 mice with P. aeruginosa PAO1 (5 106 CFU, half-lethal dose); 55 mice in the control group were concurrently administered PBS with the PAO1. We detected and analyzed: body temperature; amount of P. aeruginosa in homogenates; count of total number of nucleated cells and of mononuclear macrophages; protein concentration in bronchoalveolar lavage fluid (BALF); lung wet-to-dry weight ratio; cytokines in BALF and blood; and lung morphology. To study survival rate, concurrent administration of KGF-2 (experimental group) versus PBS (control) with a lethal dose of PAO1 (1 107 CFU was performed, and survivorship was documented for 7 days post-inoculation. The bacterial CFU in lung homogenates was significantly decreased in the KGF-2 group compared to the control group. There were significantly more mononuclear macrophages in the BALF from the KGF-2 group than from the control group (p < 0.05). KGF-2 increased the surfactant protein and GM-CSF mRNA in lung at 6 h and 72 h after inoculation. Significant reduction of lung injury scores, protein concentrations, lung wet-to-dry weight ratio, and IL-6 and TNF-a levels was noted in the KGF-2 treated rats at 72 h after inoculation (p < 0.05). The 7-day survival rate of the KGF-2 group was significantly higher than that of the control group (p < 0.05). Concurrent administration of KGF-2 facilitates the clearance of P. aeruginosa from the lungs, attenuates P. aeruginosa-induced lung injury, and extends the 7-day survival rate in mice model with P. aeruginosa pneumonia. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Keratinocyte growth factor-2 Pseudomonas aeruginosa Pneumonia Lung injury
1. Introduction Pseudomonas aeruginosa (P. aeruginosa), an opportunistic pathogen, frequently causes lower airway infection in patients with impaired immunity [1]. Antibiotic-resistant strains of P. aeruginosa are emerging and spreading in many countries. Infection with
* Corresponding author. Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Tel.: þ86 21 64041990; fax: þ86 21 64187165. ** Corresponding author. Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Tel.: þ86 21 64041990; fax: þ86 21 64187165. E-mail addresses: [email protected] (Y. Song), [email protected] (H. Li).
multidrug-resistant (MDR) and extensively drug-resistant (EDR) isolates that are insensitive to all standard antipseudomonal antibiotics (including carbapenems, fluoroquinolone and aminoglycosides) except colistin represents a special challenge, as physicians may have exhausted all effective antibiotics in treating P. aeruginosa infection [2,3]. New therapeutics, that can potently inhibit P. aeruginosa infection but do not frequently generate drug resistance, are urgently needed. Keratinocyte growth factor-2 (KGF-2), a member of the fibroblast growth factor family, is a 208 amino acid polypeptide, also known as fibroblast growth factor-10 (FGF-10) [4]. Similar to KGF, KGF-2 is mainly expressed and secreted by stromal cells; once released, it binds with high affinity to FGF receptors 2IIIb (FGFR2III-b) and 1III-b (FGFR1III-b) that are expressed exclusively on epithelial and endothelial cells [5]. KGF-2 is also similar to KGF in
http://dx.doi.org/10.1016/j.jiac.2015.10.005 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
N. Feng et al. / J Infect Chemother 22 (2016) 44e52
structure and biological function. Thus, KGF-2 acts as a paracrine mediator, facilitating epithelial cell proliferation/differentiation, angiogenesis and barrier function of capillary monolayers [6e8]. KGF-2 was found to accelerate healing of corneal epithelial wounds [9] and incisional wounds in vivo [10] and to reduce inflammatory bowel disease in a preclinical model [11]. These known biological functions of KGF-2 deem it a potential therapeutic. Recently, KGF-2 has been in phase II trials for topical treatment of chronic venous ulcers and systemic treatment of ulcerative colitis [12]. KGF-2 also prevented mucositis in patients who received chemotherapy after bone marrow transplantation [13]. Prophylactic application of KGF-2 requires further investigation. Our recent studies demonstrated that pretreatment with KGF-2 attenuated severe pulmonary edema [14] and lung injuries induced by ischemia/reperfusion [15], ventilator [16] and lipopolysaccharide [17] in rats. Viget et al. [18] showed that KGF pretreatment had antibacterial effects at the acute stage of P. aeruginosa pneumonia in rats. Wu et al. [19] also found KGF pretreatment could facilitate clearing Gram-negative pathogens from the lung within 24 h post-infection. Lee et al. [20] showed that KGF secreted by mesenchymal stem cells could kill E. coli perfused from human lungs under ex vivo culture conditions. Those results suggest that KGF-2 had great potential as a prophylactic and therapeutic, but definite effects of KGF-2 in inhibiting P. aeruginosa infection in the lungs remains to be established. The aim of this study was to determine whether concurrent administration of KGF2 with P. aeruginosa could inhibit bacterial infection and mitigate P. aeruginosa pneumonia in early (6-h) and late (24-h and 72-h) pneumonia after pathogen instillation in a mouse model. 2. Materials and methods 2.1. Animals A total of 180 female BALB/c mice, 6e8 weeks old and certified as specific pathogen-free (SLAC Laboratory Animal, China), were housed in Zhongshan Hospital Animal Care Facility. All animals had access to food and water ad libitum. The experimental protocol was approved by the Animal Care and Use Committee, Fudan University, Shanghai. 2.2. Bacteria This study used the P. aeruginosa PAO1 strain, for which the lethal dose was previously titrated 1 107 CFU (unpublished data). P. aeruginosa was incubated in tryptic soy broth (Oxide Microbiology Products, England) at 37 C; then the culture was washed twice and resuspended in sterile PBS. 2.3. Experiment grouping 2.3.1. Half-lethal dose of P. aeruginosa and KGF-2 One hundred and ten mice were randomly divided into an experimental group (intratracheal administration of P. aeruginosa and KGF-2) and a control group (intratracheal administration of P. aeruginosa and PBS). In the experimental group, recombinant human KGF-2 (rhKGF-2, 5 mg/kg in 0.05 ml; New Summit Biopharma, Shanghai, China) was administered to mice concurrently with inoculation with P. aeruginosa (5 106 CFU, half-lethal dose) by the intratracheal route. 2.3.2. Lethal dose of P. aeruginosa and KGF-2 To study the effect of KGF-2 on survival of P. aeruginosa-infected mice, the remaining 40 mice were randomly divided into a experimental group (intratracheal instillation of 1 107 CFU P. aeruginosa
45
and rhKGF-2, 5 mg/kg in 0.05 ml; n ¼ 20) and a control group (intratracheal instillation of 1 107 CFU P. aeruginosa and 0.05 ml PBS; n ¼ 20). The survival of mice in all groups was documented for 7 days after inoculation. 2.3.3. KGF-2 and PBS To study the effect of KGF-2 on the total cell number and the macrophages counts in BALF, 10 mice were randomly divided into a KGF-2þPBS group (rhKGF-2, 5 mg/kg in 0.05 ml) and PBS group (PBS,0.05 ml). 2.4. Intratracheal instillation Mice were inoculated by instilling the bacteria with KGF-2 or PBS into the left lung as previously described [21]. Briefly, following anesthesia with avertin, mice were placed on a board with their heads elevated at 45 . Then 50 ml of bacterial working solution was instilled into the left lung through the trachea using a 22G gavage needle. Mice were recovered for 15 min prior to replacement into their cages. All animals were active and appeared normal in 30 min post-inoculation. 2.5. Monitor of rectal temperature Rectal temperature was hourly monitored for initial 12 h post bacterial instillation, then daily for 7 days (5 mice for each group). 2.6. Lung and blood bacterial enumeration At 6, 24, and 72 h after inoculation, 5 mice per group in the halflethal dose experiment were euthanized with a high dosage of avertin. Blood and lungs were collected under sterile conditions. The left lung was removed and placed in 1 ml of sterile PBS and homogenized for quantification of bacteria. Serial ten-fold dilutions of lung homogenates and whole blood in sterile PBS were carried out. Then different dilutions were spread onto pseudomonas centrimide agar plates (Oxide Microbiology Products, England) for overnight incubation at 37 C prior to enumeration. 2.7. Lung wet to dry weight ratio (W/D) Mice were euthanized with averting, the left lung was isolated and the wet weight was recorded. The lungs were then placed in a 60 C incubator for 3 days, during which the dry weight was recorded to determine the wet-to-dry weight ratio. 2.8. Bronchoalveolar lavage fluid Upon euthanasia of 5 mice per group in the half-lethal dose experiment at each time point described above, bronchoalveolar lavage fluid (BALF) was also collected prior to lung removal from the left lung by cannulating the trachea and gently flushing the left lung three times with 0.5 ml PBS. BALF was immediately centrifuged at 1500 g for 10 min at 4 C, then the supernatant was stored at 80 C for further analysis, while the cell pellet was used for cell cytospin preparation. 2.9. Total cell count and differential cell count The total number of nucleated cells in BALF was counted with hemocytometer. The resuspended BALF cells were centrifuged, transferred to slides and stained with Wright'seGiemsa (Jiancheng, Nanjing, China). Finally, leukocytes and macrophages on all slides were quantified by counting a total of 200 cells/slide at 40 magnification.
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2.10. Protein concentration and cytokine levels in BALF Total amount of proteins in BALF was measured using the bicinchoninic acid protein assay kit (Thermo Scientific, Pittsburgh, PA, USA). Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) concentrations in BALF and plasma were measured by an ELISA kit (R&D Systems, USA). All procedures followed the instructions provided by each kit. 2.11. Lung histopathology In the half-lethal dose experiment, another 5 mice per group were euthanized at 6, 24, and 72 h after inoculation with a high dose of avertin. The left lung was surgically removed and fixed in 10% formalin for histology. Sections were stained with Hematoxylin and eosin (H&E). The lung injury was scored by a pathologist who was blind to sample ID [22]. 2.12. Quantitative real-time polymerase chain reaction analyses Total RNA was extracted using the Trizol (Invitrogen, CA, USA). RNA was reverse-transcribed, and real-time polymerase chain reaction (qPCR) was performed using primers for the examined transcripts (Table 1). 2.13. Statistical analysis Quantitative data was presented as mean ± SD and analyzed using SPSS v18.0 (New York, IBM, USA). Comparisons among multiple groups were performed with one-way ANOVAs, while comparisons between two groups used the ManneWhitney U test. Survival rates were plotted on KaplaneMeier product limit curves and compared by log-rank test. A p value 0.05 was considered statistically significant. 3. Results 3.1. KGF-2 alleviated lung injury in the Pseudomonas aeruginosa pneumonia Lung edema, inflammatory cell infiltration, alveolar hemorrhage and hyaline membrane formation were evident in the alveolar space at 6 h (Fig. 1A), 24 h (Fig. 1B) and 72 h (Fig. 1C) after bacterial infection in both control and KGF-2 groups. However, average lung injury score in the KGF-2 group was significantly lower than the control group at 72 h after inoculation (p < 0.05, Fig. 1D). 3.2. KGF-2 facilitated bacterial clearance from the infected lung Average body temperature in mice with PAO1-induced pneumonia was decreased in the first 6 h post-inoculation for both the
control and experimental groups; however, the body temperature of mice in the study group was significantly higher than those in the control group (p < 0.01, Fig. 2A), suggesting KGF-2 administration attenuated the decline in body temperature within 24 h of the pneumonia. Mice body temperature from both groups returned to normal over a 3-day period. Furthermore, the total number of CFUs detected in the lungs was significantly lower in the KGF-2 group at 6, 24 and 72 h after inoculation compared to the control group (p < 0.05, Fig. 2B). However, there was no viable bacteria in blood samples and the mice did not develop bacteremia.
3.3. Effect of KGF-2 on lung injury indices following intratracheal P. aeruginosa We also determined the lung injury through analyzing lung wet/ dry weight ratios. At 6 h, 12 h and 72 h post-infection, the lung wetto-dry weight ratios of left lungs from animals with untreated control group were significantly higher than those treated with KGF-2 (Fig. 3A, p < 0.05). There was evidence of significantly reduced injury (p < 0.05) in the KGF-2 group comparing the control group in all analyses including the concentration of total proteins (Fig. 3B, p < 0.05). We analyzed the number of cells in BALF. No significant difference in the total number of nucleated cells in BALF was noted between two groups (Fig. 3C). However, the number of macrophages in the KGF-2 group was significantly higher compared to the control group (p < 0.05, Fig. 3D) at 6, 24 and 72 h after inoculation.
3.4. KGF-2 reduces inflammatory process in P. aeruginosa-induced lung injury The number of neutrophils in the KGF-2 group was significantly lower at 24 and 72 h after inoculation than in the control group (p < 0.05, Fig. 4A). There was evidence of significantly reduced injury (p < 0.05) in the KGF-2 group comparing the control group in all analyses including the concentration of IL-6 in BALF at 24 and 72 h (Fig. 4B), IL-6 in plasma at 24 and 72 h (Fig. 4C), TNF-a in BALF at 24 h (Fig. 4D), and TNF-a in plasma at 24 and 72 h (Fig. 4E). 3.5. KGF-2 restores SP-A, SP-C and GM-CSF synthesis after P. aeruginosa challenge KGF-2 treatment significantly increased expression levels of SPA, surfactant protein C (SP-C) and GM-CSF in the lung compared with control group at 6 h and 72 h after inoculation (Fig. 5). KGF-2 treatment significantly increased SP-A, SP-C mRNA and GM-CSF mRNA expression compared with control group at 6 h and 72 h after inoculation (Fig. 5A, B and C).
Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Genes
Primers (50 -30 )
GenBank accession no.
Amplicon size (bp)
Surfactant protein A
F-CATAAGGAAGCCCAGGAACA R-TCAGCAAACCCTCAACAGC F-CTGATGGAGAGTCCACCGGA R-ATC ACCACGACAACGAGGAC F-CCCTGGAGTTCTGTGGTCA R-GGGTGCTGAGAGGCTGTAGA F-AGGTCGGTGTGAACGGATTTG R-GGGGTCGTTGATGGCAACA
NM_023134.4
127
NM_011359.2
125
NM_009969.4
164
NM_008084.2
92
Surfactant protein C GM-CSF GAPDH
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); granulocyte-macrophage coloy-stimulating factor (GM-CSF).
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Fig. 1. Histological changes in control and KGF-2 group (magnification 200). Hematoxylin and eosin (H&E) staining of the lung tissue in control and KGF-2 group 6 h (A), 24 h (B), and 72 h (C) after inoculation. (D) Mean lung score for each group at 6 h, 24 h and 72 h after inoculation (n ¼ 5 mice per group). The data are presented as mean ± SD. *p < 0.05.
3.6. The number of total cells and differential cells in BALF following intratracheal administration of KGF-2 Mice were treated with KGF-2 alone or PBS, and then sacrificed and lavaged, the number of cells was analyzed in BALF. The number of total cells in KGF-2þPBS group was significantly higher compared to PBS group at 6, 24 and 72 h after inoculation (p < 0.01,
Fig. 6A). The percentage of macrophages in PBS group were significantly higher than those in KGF-2þPBS group at 6 h and 72 h after inoculation (p < 0.01, Fig. 6B). The number of macrophages in the KGF-2þPBS group was significantly higher compared to the control group at 6, 24 and 72 h after inoculation (p < 0.01, Fig. 6C). The percentage of neutrophils in KGF-2þPBS group were significantly higher than those in PBS group at 6 h and 24 h after
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higher compared to PBS group at 6 and 24 after inoculation (p < 0.01, Fig. 6E). However, the number of neutrophils in PBS group were significantly higher than those in KGF-2þPBS group at 72 h after inoculation (p < 0.01, Fig. 6E). 3.7. KGF-2 improved mouse survival rate For the lethal-dose experiment, the survival rate in the KGF-2 group was significantly higher at 75% than 50% in the control group (p < 0.05, Fig. 7). 4. Discussion
Fig. 2. (A) Body temperature curves after PAO1 infection. The rectal temperature were measured and plotted at 6 h, 24 h and 72 h (n ¼ 5 mice per group). (B) The total P. aeruginosa CFUs in the lungs at 6 h, 24 h, 72 h after inoculation (n ¼ 5 mice per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01.
inoculation (p < 0.01, Fig. 6D). However, the percentage of neutrophils in PBS group were significantly higher than those in KGF2þPBS group at 72 h after inoculation (p < 0.01, Fig. 6D). The number of neutrophils in KGF-2þPBS group was significantly
The major purpose of this study was to determine if KGF-2 administration concurrent with bacterial inoculation can mitigate P. aeruginosa infection and its pathological injury in the lung of a mouse model. We found that pulmonary injury associated with mild to moderate pneumonia was attenuated at the late stage in the KGF-2 treated mice compared to the control mice. The reduction of lung injury with KGF-2 treatment was supported by significantly reduced pulmonary edema and other histological changes. KGF-2 treatment also attenuated the circulation leakage from the P. aeruginosa-infected tissues including the total BALF protein and lung wet-to-dry weight ratio, suggesting the protective effects of KGF-2 on lung epithelial barrier. Although previous study showed that KGF pretreatment can protect rats from P. aeruginosa-induced pneumonia [22]. A major concern about this treatment approach was the different kinetics between bacterial infection and the protective effects of KGF-2. The present work confirms that lung injury and inflammation were induced 6 h after the inoculation with P. aeruginosa in mice. Previous study also showed that a typical pneumonia occurs 6 h after P. aeruginosa was instilled into the lung [23]. Additionally, there were technical concerns. In this study, the protective effects of KGF-2 did not become detectable until 72 h after inoculation, which was consistent with the previous suggestion that it may take 3 days for KGF-2 to exhibit strong therapeutic effects while a minimum duration of two days is needed for KGF-2
Fig. 3. KGF-2 improves PAO1-induced protein-rich edema. (A) Lung W/D weight ratios in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. W/D ratio in control group was significantly higher than in KGF-2 group at 72 h after inoculation (B) The total protein concentration recovered in BALF. Total protein contents in control group were significantly higher than those in KGF-2 group at 6 h, 24 h and 72 h after inoculation. (C) Total white blood cell counts in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. (D) The number of macrophages in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. The number of macrophages in KGF-2 group were significantly higher than those in control group at 6 h, 24 h and 72 h after inoculation (n ¼ 5 mice per group). The data are presented as mean ± SD. *p < 0.05.
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Fig. 4. KGF-2 reduces neutrophils infiltration and inflammatory cytokines in the BALF. (A) The number of neutrophils in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. Concentrations of cytokines in BALF and plasma of the KGF-2 and control groups at 6 h, 24 h, and 72 h after inoculation (n ¼ 5 mice per group). (B) The concentration of IL-6 in BALF. (C) The concentration of IL-6 in plasma culation. (D) The concentration of TNF-a in BALF. (E) The concentration of TNF-a in plasma (n ¼ 5 mice per group). The data are shown as mean ± SD. *p < 0.05 **p < 0.01.
Fig. 5. SP-A, SP-C and GM-CSF mRNA level at 6 h and 72 h after inoculation. (A) SP-A mRNA expression in the lung. (B) SP-C mRNA expression in the lung. (C) GM-CSF mRNA expression in the lung. *p < 0.05 **p < 0.01.
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Fig. 6. The number of total cells and differential cells in BALF following intratracheal KGF-2 administration. (A) Total white blood cell counts in KGF-2þPBS group and PBS group at 6 h, 24 h and 72 h after inoculation (P < 0.01). (B) The percentage of macrophages in KGF-2þPBS and PBS group at 6 h, 24 h and 72 h after inoculation. (C) The number of macrophages in KGF-2þPBS and PBS group at 6 h, 24 h and 72 h after inoculation. (D) The percentage of neutrophils in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation. (E)The number of neutrophils in KGF-2 and control group at 6 h, 24 h and 72 h after inoculation (n ¼ 5 mice per group). The data are presented as mean ± SD. *p < 0.05 **p < 0.01 ***p < 0.001.
to reach full effect [14e17]. The mechanisms underlying the protective effects of KGF-2 in P. aeruginosa-induced acute lung injury remain incompletely understood. The present study investigated the role of KGF-2 in reducing the bacteria number and lung inflammation caused by P. aeruginosa instillation, and changes in producing surfactant proteins. We also noted that inclusion of KGF-2 in the inoculum increased bacterial clearance in the lung as observed in the early and late
Fig. 7. Survival rates of mice in control and KGF-2 groups. The 7-day survival rate was calculated and plotted following PAO1 (1 107 CFU)-induced pneumonia in the control and KGF-2 groups (n ¼ 20 mice per group). *p < 0.05.
phase of P. aeruginosa pneumonia, suggesting that local administration of KGF-2 may potentially function as an adjunct therapy to antibiotics for mild pneumonic patients infected with multidrugresistant bacteria. There are likely multiple mechanisms underlying the effect of inhibiting bacteria, though exactly what they may be remains unclear. To study the mechanisms underlying the favorable effects of KGF-2 in reducing bacterial number, we investigate the effect of KGF-2 on the total cell number, the macrophages counts and the number of neutrophils in BALF. In our study, the number of macrophages and neutrophils in the KGF-2þPBS group was significantly higher compared to the PBS group at 6, 24 and 72 h after inoculation. Ferreira et al. [24] showed that bacterial viability was reduced in the presence of KGF; however, KGF alone did not show any inhibiting effect on P. aeruginosa. Our data suggests that KGF-2 increase the number of macrophages at 6, 24 and 72 h after inoculation. Studies [16,17] examined the expression of surfactant proteins (SP) in lung tissue and found that KGF-2 pretreatment partially restored SP-A and SP-C expression. Thus we examined the SP-A and SP-C mRNA level in lung tissue and demonstrated that KGF-2 treatment partially restored SP-A and SP-C mRNA expression at early and late phases of pneumonia. The increased mRNA expression of surfactant protein might be attributed to alveolar type II
N. Feng et al. / J Infect Chemother 22 (2016) 44e52
epithelial cell proliferation and differentiation, and might indirectly prevent a permeability defect which could be a potential mechanism to deliver KGF-2 therapeutic benefits. Another study [25] showed that surfactant protein A acted as an opsonin to increase the number of macrophages in BALF and stimulate macrophage phagocytosis of a live mucoid strain of P. aeruginosa at early and late phases of pneumonia. A mechanistic investigation [19] demonstrated that KGF augmented the host defenses to E. coli and P. aeruginosa in the lungs, partly through GM-CSF-dependent macrophage recruitment and activation. Thus we also examined the GM-CSF mRNA level in lung tissue and demonstrated that KGF-2 treatment partially restored SP-A and SP-C mRNA expression. A report by Lee et al. [20] also showed that KGF increased bacterial killing through increased alveolar macrophage phagocytosis and decreased apoptosis of human monocytes by AKT phosphorylation. The inflammatory response was attenuated in late P. aeruginosa pneumonia of mice treated with KGF-2. The neutrophil counts in BALF collected in the late phase of pneumonia were significantly lower in the KGF-2 group than in the control group. Several pro-inflammation cytokines, including IL-6, TNF-a and IL1b, are involved in the initiation and development of pulmonary inflammatory response. We found that the TNF-a and IL-6 levels in BALF and blood were decreased in the KGF-2 group. KGF pretreatment reduced the blood neutrophil counts at day 3 post-injury [26] and pulmonary neutrophil counts within 8 h post-injury [27]. We previously demonstrated that a protective effect of KGF-2 pretreatment in a VILI rat model was probably mediated through decreased release of inflammatory cytokines, accompanied by reduced neutrophil infiltration and activation [16]. Thus, KGF-2 may attenuate pneumonia-associated lung inflammation by suppressing the expression and release of proinflammatory cytokines, reduces the signals attracting neutrophils to the lungs. Finally, our data suggests that the concurrent administration of KGF-2 improved survival rate of mice with lethal pneumonia. This observation was in agreement with the report that the survival was significantly increased in severe pneumonic rats with KGF pretreatment [18]. Multi-drug resistant P. aeruginosa infection is a severe problem in clinical practices associated with high mortality and morbidity [28], and the current effective treatment is very limited, thus a new therapeutic approach with promising outcomes is highly demand. One limitation of this study was that we only investigated the protective effects of concurrent administration of KGF-2 for kinetic and technical reasons. The concurrent administration of both KGF-2 and inoculum reduces the number of animal deaths because the mice easily died of repeated intratracheal intubation if KGF-2 was administered after the inoculation. A fine mixture of PAO1 and KGF2 made before the instillation can be evenly distributed to the same sites of the lung, an important requirement for minimizing procedural variation and achieving maximal effect. Our reasoning was that it would favor further testing of the therapeutic effects of KGF2 in the rat model if this study further confirmed the protection against P. aeruginosa infection. We are encouraged by our results and, in the future, will test the therapeutic effects of KGF-2 administrated after infection. In conclusion, in this study, we found that concurrent administration of KGF-2 exhibited protective effects through increased bacterial clearance and reduced lung injury in mild to moderate pneumonia. KGF-2 also increased the survival rate in mice with severe P. aeruginosa pneumonia. Our results suggest the local administration of KGF-2 may function as an adjunct therapy to antibiotics in patients with P. aeruginosa pneumonia.
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Conflict of interest The authors report no conflicts of interest. The authors are solely responsible for the contents and interpretation of the results. Acknowledgments The authors thank the members of the Bai laboratory for their invaluable advice and discussions. The KGF-2 was a gift from Xinshengyuan Pharmaceutical Company, China. The work was partly supported by Shanghai Leading Academic Discipline Project (Project Number: B115), The National Nature Science Foundation of China (811700056, 81400018, 81400002), The Shanghai Committee of Science and Technology (11411951102, 12JC1402300), and the Academic Special Science and Research Foundation for PhD Education (20130071110044). References [1] Coggan KA, Wolfgang MC. Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype. Curr Issues Mol Biol 2012;14:47e70. [2] Cabot G, Ocampo-Sosa AA, Dominguez MA, Gago JF, Juan C, Tubau F, et al. Genetic markers of widespread extensively drug-resistant Pseudomonas aeruginosa high-risk clones. Antimicrob Agents Chemother 2012;56: 6349e57. [3] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268e81. [4] Yamasaki M, Miyake A, Tagashira S, Itoh N. Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family. J Biol Chem 1996;271:15918e21. [5] Emoto H, Tagashira S, Mattei MG, Yamasaki M, Hashimoto G, Katsumata T, et al. Structure and expression of human fibroblast growth factor-10. J Biol Chem 1997;272:23191e4. [6] Ishiwata T, Naito Z, Lu YP, Kawahara K, Fujii T, Kawamoto Y, et al. Differential distribution of fibroblast growth factor (FGF)-7 and FGF-10 in L-arginineinduced acute pancreatitis. Exp Mol Pathol 2002;73:181e90. [7] Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, et al. Fgf10 is essential for limb and lung formation. Nat Genet 1999;21:138e41. [8] Igarashi M, Finch PW, Aaronson SA. Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J Biol Chem 1998;273:13230e5. [9] Wang X, Zhou X, Ma J, Tian H, Jiao Y, Zhang R, et al. Effects of keratinocyte growth factor-2 on corneal epithelial wound healing in a rabbit model of carbon dioxide laser injury. Biol Pharm Bull 2010;33:971e6. [10] Jimenez PA, Rampy MA. Keratinocyte growth factor-2 accelerates wound healing in incisional wounds. J Surg Res 1999;81:238e42. [11] Miceli R, Hubert M, Santiago G, Yao DL, Coleman TA, Huddleston KA, et al. Efficacy of keratinocyte growth factor-2 in dextran sulfate sodium-induced murine colitis. J Pharmacol Exp Ther 1999;290:464e71. [12] Robson MC, Phillips TJ, Falanga V, Odenheimer DJ, Parish LC, Jensen JL, et al. Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Repair Regen 2001;9:347e52. [13] Freytes CO, Ratanatharathorn V, Taylor C, Abboud C, Chesser N, Restrepo A, et al. Phase I/II randomized trial evaluating the safety and clinical effects of repifermin administered to reduce mucositis in patients undergoing autologous hematopoietic stem cell transplantation. Clin Cancer Res 2004;10: 8318e24. [14] She J, Goolaerts A, Shen J, Bi J, Tong L, Gao L, et al. KGF-2 targets alveolar epithelia and capillary endothelia to reduce high altitude pulmonary oedema in rats. J Cell Mol Med 2012;16:3074e84. [15] Fang X, Wang L, Shi L, Chen C, Wang Q, Bai C, et al. Protective Effects of KGF-2 on Ischemia/Reperfusion-Induced Lung Injury in Rats. Am J Respir Cell Mol Biol 2014;6:1156e65. [16] Bi J, Tong L, Zhu X, Yang D, Bai C, Song Y, et al. Keratinocyte growth factor-2 intratracheal instillation significantly attenuates ventilator-induced lung injury in rats. J Cell Mol Med 2014;18:1226e35. [17] Tong L, Bi J, Zhu X, Wang G, Liu J, Rong L, et al. Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats. Respir Physiol Neurobiol 2014;201:7e14. [18] Viget NB, Guery BP, Ader F, Neviere R, Alfandari S, Creuzy C, et al. Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2000;279:L1199e209. [19] Wu H, Suzuki T, Carey B, Trapnell BC, McCormack FX. Keratinocyte growth factor augments pulmonary innate immunity through epithelium-driven,
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