Angiotensin-(1–7) Peptide Hormone Reduces Inflammation and Pathogen Burden during Mycoplasma pneumoniae Infection in Mice

The peptide hormone, angiotensin (Ang-(1–7)), produces anti-inflammatory and protective effects by inhibiting production and expression of many cytokines and adhesion molecules that are associated with a cytokine storm. While Ang-(1–7) has been shown to reduce inflammation and airway hyperreactivity in models of asthma, little is known about the effects of Ang-(1–7) during live respiratory infections. Our studies were developed to test if Ang-(1–7) is protective in the lung against overzealous immune responses during an infection with Mycoplasma pneumonia (Mp), a common respiratory pathogen known to provoke exacerbations in asthma and COPD patients. Wild type mice were treated with infectious Mp and a subset of was given either Ang-(1–7) or peptide-free vehicle via oropharyngeal delivery within 2 h of infection. Markers of inflammation in the lung were assessed within 24 h for each set of animals. During Mycoplasma infection, one high dose of Ang-(1–7) delivered to the lungs reduced neutrophilia and Muc5ac, as well as Tnf-α and chemokines (Cxcl1) associated with acute respiratory distress syndrome (ARDS). Despite decreased inflammation, Ang-(1-7)-treated mice also had significantly lower Mp burden in their lung tissue, indicating decreased airway colonization. Ang-(1–7) also had an impact on RAW 264.7 cells, a commonly used macrophage cell line, by dose-dependently inhibiting TNF-α production while promoting Mp killing. These new findings provide additional support to the protective role(s) of Ang1-7 in controlling inflammation, which we found to be highly protective against live Mp-induced lung inflammation.

Infection with the Mp, also known as "walking pneumonia", is a common cause of respiratory illnesses in children and often leads to exacerbations in asthma and COPD patients [31][32][33][34]. Colonization of Mp in the airway is a potent inducer of TNF-α production and is associated with increased neutrophilia in the lung [31]. The objective of the present study was to determine if treatment with Ang-(1-7) peptide would provide protective and anti-inflammatory effects in Mp-challenged mice. We discovered that when given within 2 h of Mp infection, one dose of Ang-(1-7) delivered to the lungs significantly reduced neutrophilia and Muc5AC, as well as TNF-α and KC. Despite decreased inflammation, Ang-(1-7) treatment also resulted in significantly lower Mp burden in the lung tissue, which in vitro studies suggest may be through Ang-(1-7)-dependent enhanced macrophage killing mechanisms. To the authors' knowledge, this study is the first to report these findings.

Pathogen-Challenge and Drug Treatment
Mp was given via intranasal instillation at 1 × 10 8 Mp delivered in 50 µL per mouse, as previously described [35]. The experimental vehicle group, which did not receive the Mp, received a dose of sterile saline via intranasal instillation. Ang-(1-7) was given two hours post-infection via forced oropharyngeal instillation at a low dose of 0.3 mg/kg and a high dose of 1.0 mg/kg, while mice were under isoflurane anesthesia. Doses of Ang-(1-7) were based on previous publications in which Ang-(1-7) was active in repressing inflammation in murine models in the 0.3 and 1.0 mg/kg range [6,13]. Forced oropharyngeal delivery is used in place of traditional intratracheal incisions as we have previously shown [36]. All members performing this procedure have been trained with Evan's blue dye to insure their technique results in lung delivery, and not delivery to the gut. The animals that were not treated with Ang-(1-7) received sterile saline via oropharyngeal instillation. Bronchoalveolar lavage (BAL) fluid and lungs from the mice were harvested twentyfour hours post-infection for cellular analysis, qPCR, ELISA, and histological analysis of inflammatory biomarkers.

Bronchoalveolar Lavage (BAL) Fluid Cell Counts and Cell Differentiation
Twenty-four hours post-infection, mice were euthanized through intraperitoneal injection of a lethal dose of urethane. Lungs were gently flushed with 1.25 mL of PBS (0.1 mM EDTA) to obtain bronchoalveolar lavage fluid (BALF) via a cannulated trachea. The cell-free lavage fluid was used to assess cytokines and chemokines. BALF cells were enumerated by an automated cell counter (Countess, Thermo Fisher Scientific, Waltham, MA, USA) with Trypan blue exclusion for cell viability. Differential cell counts were assessed by use of the Easy III Stain Kit.

Lung Tissue Preparation for Histological Analysis
Both left and right lung lobes were collected from each mouse subject following BALF collection. Right lung lobes were collected and processed for RNA extraction. Left lung lobes were preserved by immersion in 10% formalin. Left lung lobes were transferred to immersion in 70% ethanol at least 3 days following collection of samples. Left lung samples were processed for hematoxylin and eosin (H&E) staining, and sections of each lung sample were scored in a blinded manner according to a standard scale: 0 = only alveolar macrophages were detected throughout entire tissue section; 1 = very few neutrophils observed throughout entire tissue section; 2 = neutrophils present in alveolar airspaces only; 3 = neutrophils observed in alveolar airspaces and in lymphatics; 4 = neutrophils observed in alveolar airspaces, in lymphatics and in the lumen of large airways.

RT-PCR Analysis
Right lung lobe tissue was collected from each subject and processed for RNA extraction, cDNA synthesis, and real-time polymerase chain reaction (RT-PCR) analysis. Bio-Rad TM (Bio-Rad laboratories, Hercules, CA, USA) cDNA Synthesis kit was used to synthesize DNA from 1 µg of total RNA. Quanta bio PerfeCTa SYBR Green Supermix (Quanta BioSciences, Gaithersburg, MD, USA) was used to perform RT-PCR. The gene expression of Mp P1-adhesin, TNF-α, Muc5ac, and KC were measured in mice lung tissue by RT-PCR. The mammalian housekeeper gene cyclophilin was used for expression level normalization.

Growth and Stimulation of RAW 264.7 Cells
RAW 264.7 cells were purchased from ATCC (TIB-71 ™ , Manassas, VA, USA) and grown according to standard conditions. For TNF-α stimulation, RAW cells were grown until confluent in 24-well culture plates (~500,000 cell per well). On the day of challenge, media was removed and replaced with media containing differing doses of Ang-(1-7) or vehicle control, 30 min prior to Mp stimulation. Mp was added at a MOI of 10:1 and allowed to stimulate cells for 4 h, after which the media was removed and TNF-α levels determined by ELISA. For Mp killing assays, RAW cells were seeded at a density of~100,000 cells per well into a 96-well tissue culture plate and allowed to adhere (~4 h). Media was removed and prepared Mp was added at a MOI of 2:1 (200,000 Mp to 100,000 seeded cells) with increasing doses of Ang-(1-7) or vehicle control (saline). After 18 h, a 10-µL sample was taken from each well, diluted 1:100 in SP4 media and plated on PPLO agar plates. After 2 weeks of incubation at 35 • C with no CO 2 , Mp counts were enumerated with the aid of a microscope at 4x magnification. At the dosing range used for these studies, Ang-(1-7) did not appear to directly impact Mp growth alone as no differences in colony counts were detected when Ang-(1-7) was added to Mp alone in the range of 0-0.5 µg/mL. Cell viability was assessed over a 24-h period, in which increasing doses of Ang-(1-7) were added by Trypan blue exclusion with the aid of an automated cell counter (Countess, Thermo Fisher Scientific, Waltham, MA, USA). Doses were examined in triplicate wells.

ELISA Analysis
Media from the RAW cell studies (diluted 1:10-1:100) were examined for TNF-α protein levels according to standard methods of the Mouse TNF-α ELISA MAX Deluxe Set protocol (R&D Biosystems, Minneapolis, MN, USA).

Effect of Ang-(1-7) on Inflammatory Cells in the BALF Post Mp-Infection
Infection with Mycoplasma pneumoniae resulted in a significant decrease in the percentage of macrophages, while increasing the percentage of neutrophils present in the BALF as compared to non-infected controls ( Figure 1A,C). The total number of macrophages remained consistent ( Figure 1B); however, the total number of neutrophils in the Mpinfected only group was significantly increased compared to non-infected vehicle control ( Figure 1D). The group receiving the high dose (1.0 mg/kg) Ang-(1-7) treatment following Mp infection had significantly fewer neutrophils, as measured as a percentage of cells recovered and by total neutrophil counts as compared to the Mp infection only group ( Figure 1C,D). The group receiving a low dose (0.3 mg/kg) Ang-(1-7) treatment had a significant decrease in the total number of neutrophils as compared to the group with Mp infection only ( Figure 1D).

Effect of Ang-(1-7) on Mp Burden in Lung Tissue
Colonization and infection by Mp occurs through adhesion of the bacteria to cells in the host respiratory tract [37]. Adhesin-P1, an Mp specific adhesin protein, was used to quantify the Mp burden from lung samples of the infected mice [35]. The expression of adhesin-P1 was measured by PCR. As shown in Figure 2, there was a significant decrease in Mp burden among both the low and high dose Ang-(1-7) treatment groups when compared to Mp infected only mice.

Effect of Ang-(1-7) on Muc5ac, Cytokines, and Chemokines during Mp Infection
Typical markers associated with Mp infection were assessed to determine if Ang-(1-7) would have an impact on inflammation: the mucin gene Muc5AC, the cytokine TNF-α, and the neutrophil recruiting KC (Cxcl1). Both low dose and high dose Ang-(1-7) treatments led to significantly decreased Tnf-α and Cxcl1 gene expression in lung tissue following Mp infection ( Figure 3A,B). Cxcl1 levels were elevated 12 h after Mp infection, which were significantly decreased by the high dose of Ang-(1-7). While Cxcl1 levels were lower by 24 h of infection compared to 12 h of infection, Ang-(1-7) treated groups continued to have reduced expression at both the low and high doses. Mp infection led to a slight increase in Muc5AC by 24 h; however, both Mp-infected groups treated with either low or high doses of Ang-(1-7) had significantly lower Muc5ac gene expression in lung tissue as compared to Mp-infected ( Figure 3C).

Effect of Ang-(1-7) Treatment on Lung Tissue Inflammation in Histological Sections
In order to determine if Ang-(1-7) impacted tissue inflammation during acute Mp infection, left lung sections were processed for H&E staining, and sections of each lung sample were scored according to standard scale. From the four treatment groups described above, sections of each lung sample were scored in a blinded manner according to a standard scale: 0 = only alveolar macrophages were detected throughout entire tissue section; 1 = very few neutrophils observed throughout entire tissue section; 2 = neutrophils present in alveolar airspaces only; 3 = neutrophils observed in alveolar airspaces and in lymphatics; 4 = neutrophils observed in alveolar airspaces, in lymphatics, and in the lumen of large airways. Mice treated with Ang-(1-7) during Mp infection, at both low and high doses, had significantly less lung tissue inflammation compared to Mp-infected mice given vehicle ( Figure 4).

Effect of Ang-(1-7) Treatment on Lung Tissue Inflammation in Histological Sections
In order to determine if Ang-(1-7) impacted tissue inflammation during acute Mp infection, left lung sections were processed for H&E staining, and sections of each lung sample were scored according to standard scale. From the four treatment groups described above, sections of each lung sample were scored in a blinded manner according to a standard scale: 0 = only alveolar macrophages were detected throughout entire tissue section; 1 = very few neutrophils observed throughout entire tissue section; 2 = neutrophils present in alveolar airspaces only; 3 = neutrophils observed in alveolar airspaces and in lymphatics; 4 = neutrophils observed in alveolar airspaces, in lymphatics, and in the lu-

Effect of Ang-(1-7) Treatment on Lung Tissue Inflammation in Histological Sections
In order to determine if Ang-(1-7) impacted tissue inflammation during acute Mp infection, left lung sections were processed for H&E staining, and sections of each lung sample were scored according to standard scale. From the four treatment groups described above, sections of each lung sample were scored in a blinded manner according to a standard scale: 0 = only alveolar macrophages were detected throughout entire tissue section; 1 = very few neutrophils observed throughout entire tissue section; 2 = neutrophils present in alveolar airspaces only; 3 = neutrophils observed in alveolar airspaces and in lymphatics; 4 = neutrophils observed in alveolar airspaces, in lymphatics, and in the lu- 8 high doses, had significantly less lung tissue inflammation compared to Mp-infected mice given vehicle (Figure 4).

Ang-(1-7) Dose-Dependently Inhibits TNF-α Secretion from RAW 264.7 Cells Following Mp Challenge
Since macrophages are the predominant producers of TNF-α during airway infection, we sought to determine if Ang-(1-7) would have an impact on Mp-induced TNF-α production in vitro. For these studies, we used a common macrophage cell line, RAW 264.7, which is known to produce high levels of TNF-α. As shown in Figure 5A, Ang-(1-7) had no impact on TNF-α secretion at low doses; however, starting at doses of 5 μg/mL or higher, we did see an inhibition of TNF-α by RAW 264.7 cells. We based our Ang-(1-7) dose response to be within the limits of cell tolerability, as this peptide has not shown toxicity at levels up to 1000 μg/ml in five other cell lines, as recently published [38]. In line with these results, Ang-(1-7) did not reduce RAW 264.7 cell viability at any of the doses tested over a 24 h period ( Figure 5B).  Since macrophages are the predominant producers of TNF-α during airway infection, we sought to determine if Ang-(1-7) would have an impact on Mp-induced TNF-α production in vitro. For these studies, we used a common macrophage cell line, RAW 264.7, which is known to produce high levels of TNF-α. As shown in Figure 5A, Ang-(1-7) had no impact on TNF-α secretion at low doses; however, starting at doses of 5 µg/mL or higher, we did see an inhibition of TNF-α by RAW 264.7 cells. We based our Ang-(1-7) dose response to be within the limits of cell tolerability, as this peptide has not shown toxicity at levels up to 1000 µg/mL in five other cell lines, as recently published [38]. In line with these results, Ang-(1-7) did not reduce RAW 264.7 cell viability at any of the doses tested over a 24 h period ( Figure 5B).

Ang-(1-7) Dose-Dependently Promotes Killing of Mp by RAW Cells In Vitro
We next set out to investigate if Ang-(1-7) had an impact on Mp-killing activity in RAW 264.7 cells. In our study, we compared the killing efficiency of RAW cells with and without Ang-(1-7) over an 18 h incubation period. While RAW cells effectively reduced Mp CFUs in media by 25% in 18 h, RAW cells given Ang-(1-7) had a greater reduction in Mp CFUs in a dose-dependent manner, with the lowest dose reducing 48% (* p < 0.05 versus RAW cells + vehicle) and the highest dose reducing Mp by 75% (**** p < 0.0001 versus RAW cells + vehicle) ( Figure 5C). At the dosing range used for these studies, Ang-(1-7) did not appear to directly impact Mp growth alone as no differences in colony counts were detected when Ang-(1-7) was added to Mp alone in the range of 0-0.5 µg/mL (not shown).  or vehicle control (saline) for 18 h, after which a sample was taken from each well, diluted 1:100 in SP4 media and plated on PPLO agar plates. CFUs were counted after 2 weeks of growth. n = minimum of two repeats with triplicates for each. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by the t-test for each respective dose.

Ang-(1-7) Dose-Dependently Promotes Killing of Mp by RAW Cells In Vitro
We next set out to investigate if Ang-(1-7) had an impact on Mp-killing activity in RAW 264.7 cells. In our study, we compared the killing efficiency of RAW cells with and  1-7). Cell-free supernatants were assessed for TNF-α by ELISA. n = minimum of two repeats with triplicates for each. (B) RAW cells were incubated with increasing doses of Ang-(1-7) in triplicate for 24 h and viability assessed by Trypan blue exclusion using a Countess cell counter. (C) RAW cells were stimulated with Mp (MOI of 2:1) with increasing doses of Ang-(1-7) or vehicle control (saline) for 18 h, after which a sample was taken from each well, diluted 1:100 in SP4 media and plated on PPLO agar plates. CFUs were counted after 2 weeks of growth. n = minimum of two repeats with triplicates for each. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by the t-test for each respective dose.

Discussion
The role of Ang-(1-7) in reducing allergic inflammation in animal models has been previously reported; however, the role of this protective peptide hormone in treating infections common among many chronic asthma sufferers has not been described. Mycoplasma pneumoniae is known to colonize the airways of patients considered to have chronic asthma, and this underlying bacterial infection is thought to contribute to asthma exacerbations among these patients. Therefore, we thought it would be important to understand how secondary illnesses common among asthmatics affect the efficacy of a potentially new drug being considered for the treatment of asthma.
Our research aimed to address how Ang-(1-7) would perform during acute infection with Mp. The main findings of this study indicate that Ang-(1-7) has the ability to provide protection in an experimental mouse model of acute Mp infection by ameliorating inflammatory phenotypes within 24 h of infection. We chose to test Ang-(1-7) delivery to the lungs, as we believed this would have the greatest impact to controlling inflammation. However, additional therapeutic routes, including oral or aerosol delivery via nebulizer, which would be congruent with options for humans, should be explored. In support of this, our colleagues recently reported excellent aerosol dispersion performance of Ang-(1-7) with a human DPI device and in vitro human cell viability assays showed that Ang-(1-7) was biocompatible and safe for different human respiratory cells [38]. We chose to only assess Ang-(1-7) treatment when given shortly (within 2 h) after an infectious challenge. While our findings indicate that high doses of Ang-(1-7) led to decreased infiltrating neutrophils, as well as the cytokines and chemokines that can recruit them, TNF-α and Cxcl1, we recognize in humans it would be more translatable to give Ang-(1-7) treatment after symptoms of "walking pneumoniae" are more evident, which could occur several days after exposure. Future studies should address the timing for Ang-(1-7) dosing to see how long after Mp infection, delivery of Ang-(1-7) is still efficacious to increase the availability in clinical practices.
Mp infections among asthmatic patients have been associated with a dramatic spike in levels of TNF-α [39]. We provide evidence that Ang-(1-7) treatment can significantly reduce levels of TNF-α at the RNA and protein levels during Mp infection. In addition, Ang-(1-7) dose-dependently reduced TNF-α production or secretion from Mp-stimulated RAW 264.7 cells, which are a macrophage cell line. While RAW 264.7 cells are well utilized in the field for drug screening, future studies should test Ang-(1-7) on primary macrophage populations from humans to validate our findings. Since macrophages are the predominant producers of TNF-α during respiratory infection, it is relevant that Ang-(1-7) can work directly on these cells during Mp stimulation. Reducing any downstream impacts of TNF-α driven inflammation would likely have additional positive impacts for asthmatic patients beyond those we detected and should be examined in future studies.
Similarly, mucus production is an important factor when evaluating treatments for asthma, as mucus hyper-secretion into the airway lumen obstructs airways and worsens asthma symptoms [40]. Along these lines, we found that Muc5AC gene expression was significantly decreased during Mp infection following treatment with Ang- (1-7). Interestingly, the level of Muc5AC was repressed by Ang-(1-7) during infection below the levels normally present without infection. This suggests that Ang-(1-7) may have an inhibitory impact of on mucin production in the absence of infection, which should be further studied. While an increase in mucin production was not evident at this early timepoint, future directions should examine the impact of Ang-(1-7) treatment in longer models of Mp infection to see if the reduced Muc5AC gene expression results in deceased mucin production in lung sections. Examination of lung histological sections revealed enhanced neutrophilia in airways of Mp infected mice, while those treated with Ang-(1-7) were more similar to untreated controls. So not only did Ang-(1-7) results in fewer neutrophils migrating into the lung lumen, it also impacted neutrophils migrating into the lung tissue.
Along with providing a promising therapeutic option for the treatment of asthma, the results of this study also indicate that Ang-(1-7) treatment may be helpful for controlling inflammation during Mp infection, and in the clearance of Mp. While fewer neutrophils were detected in the Ang-(1-7) treatment groups, the Mp burden was also decreased. This was somewhat surprising given that neutrophils would contribute to Mp killing in the lungs. This finding of fewer neutrophils and reduced Mp burden, suggest that another compensatory mechanism for pathogen removal may have been activated. Those could include more activation of macrophages prone for pathogen killing or an induction of epithelial-driven antimicrobial host defense.
We next conducted studies to see if Ang-(1-7) had any direct antimicrobial impact on Mp and we found that Ang-(1-7) alone was not able to reduce Mp CFUs over a two-week growth period (data not shown). However, Mp-killing studies in RAW 264.7 cells in the presence or absence of increasing concentrations of Ang-(1-7) indicated that RAW cells had increased Mp-killing efficiency with increases doses of Ang- (1-7). This suggests that Ang-(1-7) may have a therapeutic potential in stimulating macrophages to better eliminate pathogens in the lungs. Since Ang-(1-7) and analogs are known to reduce ROS and promote NO production, it is likely this Ang-(1-7) activity contributes to the enhanced Mp-killing function by macrophages. Future studies should evaluate this possible mechanism and the ability of Ang-(1-7) to assist in clearance of other infectious agents common among asthmatics, such as human rhinovirus (HRV) and respiratory syncytial virus (RSV) [41].
While most commonly known as a vasodilator agent with important roles in the cardiovascular system, Ang-(1-7) has also emerged with anti-oxidant, anti-inflammatory, and anti-fibrotic effects in several model systems [6,42]. Importantly, Ang1-7 inhibits production and expression of many cytokines and adhesion molecules associated with a cytokine storm [43,44] and delivery of exogenous Ang1-7 has been shown to improve oxygenation and can be safely delivered to humans [25,45,46]. In line with these studies, we add that not only does Ang-(1-7) reduce inflammation during live respiratory infection with Mp, it also leads to a significant reduction in Mp pathogen burden and reduction of mucin production in the airways.

Conclusions
To our knowledge, this is the first study to demonstrate the effects of Ang (1-7) peptide in Mp lung infection and offer mechanistic insight into macrophage killing mechanisms. Taken with the many previous studies in various models in which Ang-(1-7) peptide has anti-inflammatory activity, our studies not only support these findings, but also bring to light a potentially novel indication, i.e., as a therapeutic for the treatment of respiratory infections.