Angiotensin-(1-7) treatment improves pneumonia and prevents sepsis caused by pneumococcal infection

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This study investigates the therapeutic potential of Angiotensin-(1–7) [Ang-(1–7)], a peptide with anti-inflammatory and pro-resolving effects, in a murine model of pneumococcal pneumonia. In mice infected with S. pneumoniae , Ang-(1–7) reduced leukocyte infiltration, pulmonary edema, and tissue damage, and lowed production of the pro-inflammatory cytokines TNF-α, IL-6, and CXCL-1. The treatment improved bacterial clearance in the bronchoalveolar lavage and blood and increased survival rates. Importantly, when combined with the antibiotic ceftriaxone, Ang-(1–7) enhanced survival even when treatment started late in this model of pneumococcal pneumonia. Mechanistically, Ang-(1–7) enhanced the phagocytic activity of S. pneumoniae by bone marrow-derived macrophages and increased the expression of genes associated with lung barrier integrity. These results show that treatment with Ang-(1–7) decreases inflammation and improves outcomes in severe and invasive pneumococcal pneumonia, especially when combined with antibiotics, suggesting it may be a useful adjuvant therapeutic strategy in this infectious condition. Streptococcus pneumoniae Angiotensin-(1–7) inflammation sepsis phagocytosis infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Streptococcus pneumoniae is the main cause of community-acquired pneumonia (CAP) irrespective of the severity [ 1 ]. Resistance of S. pneumoniae to antibiotics is increasing in many parts of the world, an observation that makes CAP an infectious disease of significant interest. In addition, CAP represents a substantial clinical and economic burden on society [ 2 – 4 ]. Among pneumococci, serotype 3 is commonly associated with invasive and lethal infections [ 5 ]. The pathogenesis of this infection includes an important virulence factor, the capsule surrounding S. pneumoniae , which helps the bacteria escape from phagocytosis and induce intense inflammatory responses [ 6 ]. This inflammation triggered by pneumococcal infections is essential for bacterial clearance; however, it must be tightly regulated to preserve alveolar structure, prevent bacterial dissemination from the lungs to the bloodstream, and ultimately avoid fatal outcomes [ 7 ]. Bacterial dissemination is a frequent occurrence in patients with pneumonia, often contributing to extrapulmonary manifestations, including the development of sepsis [ 8 ]. The production of pro-resolving molecules during inflammation is crucial to ensure the timely resolution of the immune response and to promote the proper return of the affected tissues to homeostasis after infection [ 9 – 12 ]. Therefore, pro-resolving molecules may be useful in the context of pulmonary infections by limiting lung inflammation without impairing the host defenses against S. pneumoniae [ 13 – 17 ]. Angiotensin-(1–7) [Ang-(1–7)] is a hormone within the Renin-Angiotensin System (RAS) that recognized for its anti-inflammatory, anti-fibrotic and pro-resolving effect in various acute and chronic models of inflammatory disease [ 18 – 23 ] and, more recently, in infection models [ 24 – 26 ]. The effects of Ang-(1–7) during pneumonia and sepsis caused by S. pneumoniae has not been evaluated. Material and Methods Study design, ethical statement and mice This work was randomized, blinded and controlled by vehicle group during the experimental and analysis stages. All experimental procedures described here were conducted according to Brazilian guidelines on animal work and approved by the local Animal Ethics Committee from Universidade Federal de Minas Gerais (CETEA - UFMG - protocol number 65/2021). C57BL/6JUnib mice aged 8 to 11 weeks were maintained in a controlled environment with free access to filtered water and standard laboratory chow ad libitum . Streptococcus pneumoniae ATCC 6303 serotype 3 was grown in culture medium containing on 3.7% Todd Hewitt, 0.5% yeast extract, 5% blood, and 1.5% agar for 12 h at 37°C and 5% CO 2 . Colonies grown on plates were picked and inoculated into Todd-Hewitt broth with 0.5% yeast extract at an initial absorbance (optical density at 600 nm = 0.1) and grown to logarithmic phase (optical density at 600 nm = 0.4). S. pneumoniae was then centrifuged at 2,000 rpm for 20 min. Inoculum for infections were prepared after dilution in 0.9% saline as described previously [ 27 ]. In all experiments, the inoculum was confirmed by plating of bacterial suspension. Experimental procedure Anesthetic solution of xylazine (10 mg/Kg) and ketamine (70 mg/Kg) was administered subcutaneously to the mice. Pneumococcal infection was performed by intranasal instillation with an inoculum of 40 µL of S. pneumoniae containing 5 x 10 4 or 5 x 10 5 CFU and not infected (NI) mice received 40 µL of sterile saline by intranasal instillation. All mice were monitored until complete recovery from anesthesia. The oral formulation of Ang-(1–7) was administered via gavage every 12 hours, from 12 to 36 hours following pneumococcal infection. Mice were euthanized 48h after pneumococcal infection to assess the inflammatory response. Broncho alveolar lavage fluid (BALF) was collected to evaluate leukocyte recruitment, cytokine and chemokine production, bacterial loads and protein extravasation. Blood was collected to assess the presence of sepsis and lungs were harvested for histology, myeloperoxidase (MPO) assessment and quantification of tight junction complex expression genes. In lethality experiment, mice weights were followed for 10 days and those who reached 75% of the initial body weight were euthanized. Based on what happens with septic patients, the intraperitoneal (i.p.) ceftriaxone antibiotic (ATB) was associated with Ang-(1–7) to enhance the effect against sepsis. For oral administration, the pneumococcal infected groups received vehicle (Veh − 92 µg/Kg of HPβCD in filtered water, 100 µL, by gavage) and Ang-(1–7) group received Ang-(1–7) / HPβCD [60 µg/Kg of Ang-(1–7) and 92 µg/Kg of HPβCD in filtered water, 100 µl/ mouse, by gavage]. The control non infected group received 100 µl/ mouse of filtered water. The dose used was based on previous studies using murine models of flu [ 25 ], asthma [ 28 ], arthritis [ 29 ], emphysema [ 30 ] and pulmonary fibrosis [ 23 ]. For intraperitoneal administration, groups infected with S. pneumoniae , both Veh and Ang-(1–7), received or not ATB (10 mg/Kg in sterile saline, 100 µL) as previously described [ 31 ]. Bronchoalveolar lavage fluid (BALF), tissue extraction and total/differential cell count BALF, tissue extraction and cell count were obtained as previously described [ 25 ]. For the BALF, two aliquots of 1mL of PBS were flushed three times into the lungs, through a 1.7mm catheter inserted into the trachea to collect leukocytes and S. pneumoniae from the alveoli of mice. Thereafter, the left lung was collected and embedded in neutral buffered formalin (10%) for histological analysis and the right lobe was divided for PCR and MPO analyses. Then, BALF was serially diluted and plated in blood agar for bacterial counts (the same was done with blood). Then, BALF samples (2 mL each mouse) were centrifuged at 600 x g for 10 minutes at 4 o C. Total number of leukocytes was determined by counting leukocytes in a Neubauer chamber. For the differential count, the percentage of each leukocyte (mononuclear and polymorphonuclear) was based on morphological criteria after staining with May– Grünwald –Giemsa of slides obtained from cytospin (Shandon III) preparations. Each slide was counted three times, and the percentage was used to calculate the absolute number of each leukocyte type. In addition, BALF supernatants were used for cytokine evaluation by ELISA (R&D Systems, Minneapolis, MN, USA) and total protein quantification using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Bradford assay Pulmonary edema was assessed by quantifying total protein concentration in BALF. Protein levels were measured using a colorimetric assay, with bovine serum albumin (BSA) as the standard curve ranged from 0.125 to 2 mg/mL. After adding 2 µL of the standard curve and samples to the 96-well plate, 200 µL of Protein Assay Dye Reagent Concentrate (Bio-Rad) diluted 5x was added for detection. The reaction was measured in a spectrophotometer (BioTek Epoch – Agilent Technologies) at an absorbance read at 590 nm. ELISA assay Concentrations of TNF-α, IL-6, CCL-2 and CXCL-1 were measured in the BALF supernatants by ELISA DuoSet kits according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA). Briefly, NuncFlat 96-well plates were sensitized with specific detection antibodies diluted in PBS and incubated overnight. Then the plates were washed with 0.1% (v/v) Tween, blocked with 1% (v/v) bovine serum albumin (BSA) for 2 h and washed again after blocking. 100 µL of samples, standards and blank were added to the plates and incubated overnight. The next day, the plates were washed again and detection antibodies diluted in PBS were added. Another round of washing was performed, and streptavidin solution was added to the wells. OPD and oxygen peroxide were added to the plates for 20 min to generate a colorimetric reaction. The reaction was measured in a spectrophotometer (BioTek Epoch – Agilent Technologies) at 490 nm. Myeloperoxidase assay - MPO The right lung of mice was collected for indirect quantification of neutrophils recruitment into the tissue (MPO – myeloperoxidase assay). As previously described [ 32 ], 50 mg of lung tissue were homogenized in a buffered solution containing antiproteases. MPO levels were performed using 25 µL of the supernatant of the homogenized and 25 µL of a solution of 3,39 − 5,59-tetramethylbenzidine (1.6 mM, TMB; Sigma – dissolved in dimethyl sulfoxide) and 100 µL of H 2 O 2 (0.01 mM) diluted in phosphate buffer (pH 5.4) containing HETAB. Bone Marrow-Derived Macrophages (BMDMs) isolation BMDM were obtained as previously described [ 33 ]. Briefly, following euthanasia, tibias and femurs were collected, and bone marrow cells were flushed out using sterile medium for subsequent differentiation into macrophages. The cell suspension obtained was then centrifuged at 1200 RPM for 5 min. The pellet was resuspended in complete conditioned media for BMDM differentiation (RPMI containing 10% heat-inactivated fetal bovine serum and 30% L929 cell-conditioned medium), seeded on petri dishes and incubated at 37°C and 5% CO 2 . After 7 days, the supernatant was discarded, and adherent macrophages were detached by using a cell scraper and plated (2 x 10 5 cells/well) in 96-well plates for phagocytosis assay. Phagocytosis assay Phagocytosis was evaluated as previously described [ 24 , 26 ]. Briefly, 2 x 10 5 BMDMs were plated and incubated for 3 h for adhesion at 37°C and 5% CO 2 . Then, treatments Ang-(1–7) (152 µg/Kg of Ang-(1–7) / HPβCD) or Veh (92 µg/Kg of HPβCD) was added to the well for 18 h and then a MOI of 10 of S. pneumoniae was added to this culture of BMDMs for 3 h to allow phagocytosis (1 h of adhesion at 4°C followed by 2 h at 37°C and 5% CO 2 ). Noninternalized bacteria were washed out with 30 µg/mL of penicillin and streptomycin for 30 min. Then, BMDMs were briefly lysed with cold water and then plated on blood agar for subsequent CFU counts. Gene expression Gene expression was obtained as previously described [ 14 ]. 50 mg of the right lung lobe was collected and stored at -80°C until use. Total RNA was isolated from this tissue incubated and minced in 500 µL TRIzol (Invitrogen). Then, 200 µL of chloroform was added, and the samples were vortexed for 15 s before being centrifuged at 12,000 g for 15 min at 4°C. Aqueous phase samples were collected and 500 µL isopropanol was added to them. Samples were incubated on ice for 10 min and then were centrifuged at 12,000 g for 15 min at 4°C. The supernatants were discarded, and the pellet was washed with 75% ethanol solution. The pellet was dried at room temperature and then resuspended with 20 µL of injection water. RNA concentration was measured by nanodrop and then stored at -80°C. For cDNA preparation, samples were treated with DNAse and RNA template was converted to cDNA by reverse transcriptase using Superscript Transcriptase (Promega), according to the manufacturer’s instructions. cDNA was stored at -20°C until quantitative PCR (q-PCR) assays. qPCR was performed Sybr Green PCR Master Mix (Applied Biosystems) with addition of forward and reverse primers and samples. Runs were performed on the 7500 Fast System. Tight junction primers were designed to amplify exon-exon junctions in mRNA and the relative level of gene expression was determined by the 2^(-ΔΔ Ct) method. Data for each sample were normalized to GAPDH mRNA and expressed as relative expression. The primers used were epithelial barrier genes m-Cldn3 FWD 5′-CCAACTGCGTACAAGACGAG-3′; m-Cldn3 REV 5′-TCTTGGTGGGTGCATACTTG-3′; m-Cldn5 FWD 5′-TGGAACGCTCAGATTTCATC-3′ and m-Cldn5 REV AGGAAGGCAACCCCTCTAAG-3′. Histological analysis After the mice's left lungs were fixed in formalin solution, they were gradually dehydrated in ethanol and processed as described [ 34 ]. 4 µm sections of the tissue were stained with Hematoxylin & Eosin and analyzed by a pathologist blinded to the experiment. The score of inflammation totaled 18 points and were distributed when evaluating airway inflammation (up to 4 points), vascular inflammation (up to 4 points), parenchymal inflammation (up to 5 points) and general neutrophil infiltration in the lungs (up to 5 points). Statistical analysis Statistics were performed using GraphPad Prism 9.0. Before comparing groups, the Shapiro-Wilk normality test was performed. Then, one-way ANOVA followed by Turkey’s multiple comparisons test was used to compare more than two groups, and the unpaired t-test was used for comparisons between two groups. Survival curves were analyzed by the Long-rank test. Results with P < 0.05 were considered statistically significant. Results Administration of Angiotensin-(1–7) reduced leukocyte infiltration, improved edema formation and lung damage after pneumococcal infection The first experiments evaluated the effects of Ang-(1–7) treatment in pneumonia caused by S. pneumoniae . Mice were infected intranasally with 5x10 4 CFU and then treated with Ang-(1–7) or Veh from 12 to 36 h after pneumococcal infection. Animals were euthanized 48 h after infection (Fig. 1 a). Ang-(1–7) treatment reduced the total number of leukocyte and neutrophils in BALF at 48 h, as compared to Veh-treated animals (Fig. 1 b, c). There was no difference in mononuclear cell recruitment between mice treated with Ang-(1–7) and Veh in the airways after pneumococcal infection (Fig. 1 d). In addition, MPO activity in the lungs of mice treated with Ang-(1–7) tended to decrease when compared to Veh (Fig. 1 e). Pulmonary edema, measured by the concentration of proteins that leaked into the airways, was lower in mice treated with Ang-(1–7) than Veh (Fig. 1 f). Consistent with the control of neutrophil and leukocyte recruitment and reduction in pulmonary edema, evaluation of lung tissue injury in histopathological sections show that damage was lower in mice treated with Ang-(1–7) than Veh after pneumococcal infection (Fig. 1 g). These results suggest reduced inflammatory parameters when mice are treated with Ang-(1–7) after pneumococcal infection. Levels of pro-inflammatory cytokines and chemokines are decreased in the airways of Angiotensin-(1–7)-treated mice after pneumococcal infection Pro-inflammatory cytokines and chemokines that remain persistent and contribute to robust acute inflammation are also associated with tissue damage, morbidity and, consequently, death. Thus, Ang-(1–7) treatment, compared to Veh, has been shown to reduce CXCL-1 levels (Fig. 2 a), consistent with the lower neutrophil influx observed. Furthermore, compared to Veh, Ang-(1–7) treatment reduced CCL-2 and IL-6 (Fig. 2 b, c, respectively), which are important in the control of mononuclear cells and acute inflammation. In addition, Ang-(1–7) treatment reduced TNF-α (Fig. 2 d) associated with the lower tissue damage demonstrated. IL-10, which has an anti-inflammatory role, remained the same between the different groups analyzed (Fig. 2 e). Overall, we showed that Ang-(1–7) treatment was able to reduce the levels of pro-inflammatory mediators, contributing to a finely regulated acute inflammation. Administration of Angiotensin-(1–7) improved the survival of mice after Streptococcus pneumoniae infection To evaluate the effects of Ang-(1–7) treatment on the lethality rates, we infected mice with S. pneumoniae , treated them with Ang-(1–7) or Veh every 12 hours from 12 to 36 hours after infection. Mice were monitored the mice daily for 10 days to assess the survival rate (Fig. 3 a). We were able to observe that Ang-(1–7) treatment increased survival when compared to Veh after pneumococcal infection (Fig. 3 b). These data confirmed that controlling inflammatory parameters and tissue damage results in reduction of deaths caused by S. pneumoniae infection. Angiotensin-(1–7) treatment reduced colony forming unit (CFU) in the airways and prevented sepsis by pneumococcal infection A finely regulated immune response may contribute to more effective bacterial clearance [ 35 ]. When comparing Veh and Ang-(1–7) treatments, we observed that Ang-(1–7) not only prevented mortality but also finely modulated the immune response. We were also able to observe that this was associated with better control of bacteria replication, as seen by the lower number of S. pneumoniae in the airways and blood (Fig. 4 a, b, respectively). Therefore, although lung inflammation was greatly reduced in mice treated with Ang-(1–7), the level of inflammation observed was sufficient to control S. pneumoniae numbers in the alveoli and prevent dissemination to blood. Angiotensin-(1–7) treatment acted on bone marrow derived macrophages (BMDMs) to improve phagocytosis of Streptococcus pneumoniae The lower amount of S. pneumoniae in the airways and blood may be due to the improved ability of the cells to deal with this pathogen or may be due to a direct antibacterial action of the molecule. First, we evaluated the effect of treatment of Ang-(1–7) in BMDMs infected with S. pneumoniae . To this end, BMDMs were treated with Veh or Ang-(1–7) for 18 h, infected with a MOI of 10 of S. pneumoniae for 2 h and then plated for a further 3 h (Fig. 5 a). Treatment with Ang-(1–7) clearly increased the phagocytosis of S. pneumoniae by BMDMs (Fig. 5 b). In contrast, there was no direct effect of Ang-(1–7) on S. pneumoniae in vitro , as the bacterium maintained its usual growth phases in the groups treated with Veh or Ang-(1–7) (Supplementary Fig. 1). Thus, Ang-(1–7) acts on BMDMs and improves their phagocytic capacity, but has no direct bactericidal, bacteriostatic or even bacteriolytic action. Administration of Angiotensin-(1–7) promoted increased expression of tight junction genes in lung tissue after pneumococcal infection During bacterial infections, uncontrolled bacterial replication can cause loss of the epithelial barrier, allowing the pathogen to leak into the bloodstream and cause sepsis [ 36 , 37 ]. To evaluate the epithelial barrier, we assessed the effects of treatment with Ang-(1–7) on the expression of genes ( Cldn3 and Cldn5 ) associated with the tight junction complex. Treatment with Ang-(1–7) was associated with an increase in the expression of Cldn3 and Cldn5 in the lungs (Fig. 6 a, b, respectively), suggesting lower S. pneumoniae dissemination may have been secondary to greater epithelial barrier integrity. Combining the antibiotic ceftriaxone and Angiotensin-(1–7) as treatments in cases of severe pneumonia caused by Streptococcus pneumoniae resulted in even greater survival In cases of severe pneumonia, sepsis may occur and is associated with a significant increase in mortality. Providing adequate antibiotic treatment is crucial but is often started too late in the course of disease at a time it is no possible to reverse the septic condition [ 38 , 39 ]. In attempt to model this situation in our animal model of infection, mice were infected intranasally with a higher inoculum of S. pneumoniae (5x10 5 CFU) and then treated with Ang-(1–7) in the absence or presence of and antibiotic (ceftriaxone) or Veh from 60 to 72 h after infection (Fig. 7 a). Using this inoculum, 100% of animals were dead by day 6 after infection. Using this higher inoculum and a more delayed initiation of treatment, Ang-(1–7) alone was not associated with enhanced survival. Similarly, delayed start of Ceftriaxone treatment alone was not associated with significantly enhanced survival (Fig. 7 b). In contrast, delayed start of treatment with a combination of Ang-(1–7) and ceftriaxone greatly prevented death in this model of severe and disseminated pneumococcal infection. Discussion Pneumonia caused by Streptococcus pneumoniae remains a significant global health threat. According to the World Health Organization (WHO), in 2008, it was estimated that 541,000 children under the age of five died from pneumococcal diseases, with pneumonia being the primary manifestation. Mortality rates from pneumococcal infections can be as high as 20% in cases of severe pneumonia accompanied by sepsis [ 40 ]. The severity of pneumococcal pneumonia is largely driven by the inflammatory response it triggers. Exaggerated inflammation, if not properly regulated, can lead to excessive tissue damage and worsen the infection. In our study, we demonstrated that (i) exogenous administration of Ang-(1–7) significantly reduced leukocyte infiltration, alleviated pulmonary edema, decreased TNF-α levels, while treatment enhanced bacterial clearance and improved both survival rates. Importantly, (ii) in a model of severe and invasive pneumococcal infection, a critical and often fatal complication in hospitalized patients, Ang-(1–7) exhibited a synergistic effect when combined with antibiotics, resulting in increased survival. Mechanistically, (iii) treatment with Ang-(1–7) enhanced phagocytosis of bacteria, an effect associated with decreased bacterial loads in the lung, and enhanced epithelial cell integrity, an effect associated with decreased bacterial loads in blood. Ang-(1–7) is an endogenous heptapeptide part of the counter-regulatory branch of the Renin-Angiotensin System (RAS) and whose bioactivity extends far beyond the cardiovascular system [ 41 ]. Our studies have clearly demonstrated that Ang-(1–7) has anti-inflammatory and pro-resolving effects in vivo It has been reported to reduce lung inflammation, fibrosis, pulmonary arterial hypertension and prevent secondary pneumococcus infection after influenza [ 17 , 18 , 23 , 25 , 42 ]. The findings that administration of Ang-(1–7) makes the course of pneumococcal disease less severe, as seen by the reduction of leukocyte infiltration, mainly neutrophils in the pulmonary alveoli are consistent with the anti-inflammatory and pro-resolving actions of Ang-(1–7) in several systems [ 17 , 20 – 23 , 25 , 28 , 29 , 42 ]. They are also consistent with the effects of other pro-resolving molecules in models of pneumonia in which protection was associated with decreased neutrophil recruitment and activation [ 14 , 17 , 43 – 45 ]. In contrast, the course of the disease was worse in mice that had their neutrophils depleted [ 25 , 46 – 48 ]. Therefore, it is clear that a certain level of neutrophil recruitment and activation is necessary in the context of pneumococcal and other bacterial infections. However, excessive neutrophil influx and activation may be detrimental to the host and lead to pulmonary injury and death [ 49 ]. We were also able to observe less pulmonary edema and lower amounts of TNF-α, which are closely linked to more intact lung tissue, demonstrating less neutrophil infiltration, less inflammation of the parenchyma, vascular and airways after therapeutic administration of Ang-(1–7). Interestingly, absence of pro-resolution agents leads to an intense increase in TNF-α, causing endotoxic shock in a model of challenge with LPS [ 50 ]. This exacerbated increase in TNF-α also contributes to the pathogenesis and development of pulmonary edema [ 51 ] and causes multiple inflammatory disorders [ 52 ]. Pulmonary edema is a significant medical problem worldwide and can be life-threatening because it is related to important clinical manifestations such as shock, diffuse alveolar damage and lung hypersensitivity states [ 51 ]. Therefore, controlling TNF-α levels, reducing histopathological damage and pulmonary edema contribute to better outcomes related to pneumonia and sepsis due to S. pneumoniae . Not only TNF-α, but other cytokines, such as IL-10 and IL-6, are related to severe clinical signs of pneumonia caused by S. pneumoniae [ 53 – 55 ] and predict severe progression of the disease [ 56 ]. In addition, these levels are reduced after antibiotic treatment [ 57 ]. In the same way, it was possible to see a reduction in IL-6 and TNF-α, after the administration of Ang-(1–7) treatment. With these various inflammatory parameters limited due to Ang-(1–7) administration, there was consequently greater survival after pneumococcal infection. Reduction of severity of pneumonia is associated with reduced pulmonary injury, reduced bacterial dissemination (sepsis), and fewer early deaths [ 55 , 58 ]. We have previously shown that Ang-(1–7) was also highly protective against severe primary Influenza A virus (IAV) infection and protected against secondary S. pneumoniae infection in the lung leading [ 25 ]. Other pro-resolving molecules have been shown to be protective in models of pulmonary infection [ 14 , 17 , 43 – 45 ]. For example, the pro-resolving molecule Annexin A1 (AnxA1) also prevented excessive lung damage and the absence of AnxA1 increased lethality after pneumococcal infection [ 14 ]. Pro-resolving molecules are also effective in systemic sepsis from a source other than the lung. Indeed, administration of pro-resolving plasminogen/plasmin (Plg/Pla) protected mice from sepsis-induced by cecal ligation and puncture (CLP) lethality and enhanced the protective effect of antibiotics [ 59 ]. Reduced mortality was clearly associated with decreased pulmonary inflammation, edema, and tissue damage, as assessed histologically. Improved survival was also linked with greater control of bacterial replication in the lungs and reduced dissemination into the bloodstream, indicating decreased progression to sepsis. A reduction in bacterial load in both the lungs and bloodstream may have clearly contributed to the decreased severity of pneumococcal pneumonia and sepsis. It is important to note that Ang-(1–7) had no direct antimicrobial impact on S. pneumoniae . However, Ang-(1–7) greatly enhanced the ability of macrophages to phagocytose S. pneumoniae . Indeed, we have previously shown that Ang-(1–7) acts as a phagocyte attractant and increased the ability of BMDMs to phagocytose Escherichia coli [ 26 ]. In experimental Type 2 Diabetes Mellitus, Ang-(1–7) rescued the ability of mice neutrophils to phagocytose Staphylococcus aureus , a bacterium that also causes lung infection [ 60 ]. Additionally, in a model of Mycoplasma pneumonia infection, Ang-(1–7) demonstrated efficacy in decreasing colonization of this pathogen, known to provoke exacerbations in asthma and chronic obstructive pulmonary disease (COPD) patients, in the airways [ 24 ]. Therefore, the unique capacity of Ang-(1–7) to enhance phagocytosis of bacteria by macrophages was associated with decreased number of bacteria in the lungs and these effects may have contributed to the ability of Ang-(1–7) to decreased severity and deaths in this model of infection. S. pneumoniae may replicate in the lungs and disseminate to other tissues, causing sepsis and high degree of lethality [ 61 ]. In this regard, not only the function of leukocytes undergoing phagocytosis, but also other factors such as lung epithelial barrier integrity may be important to prevent the occurrence of dissemination and sepsis [ 62 ]. Greater integrity of the epithelial barrier may also contribute to lower lung injury and edema contributing to lower fatalities after infection [ 63 ]. Our results show that the gene expression of claudins 3 and 5, which are part of the tight junction complex (TJC) and hence relevant for epithelial barrier integrity, was increased after Ang-(1–7) administration. The reduction in IL-6 may have contributed to the increased expression of Cldn3 and Cldn 5 in the lung after Ang-(1–7) treatment, since there is an inverse correlation of claudins expression and IL-6 production [ 64 – 66 ]. In another study, the absence of pro-resolving AnxA1 impaired the upregulation of Cldn3 and Cldn5 genes, which might have accounted for the loss of lung barrier integrity during pneumococcal infection [ 14 ]. Therefore, the upregulation of these tight junction-associated genes may have contributed to the beneficial effects of Ang-(1–7) by limiting edema formation and bacterial dissemination into the bloodstream. Finally, our results showed that delayed administration of Ang-(1–7) was able to rescue the ability of antibiotics to preventing death after severe pulmonary pneumococcal infection. This is indeed very clinically relevant as both Ang-(1–7) and the antibiotic were given late in the course of infection when neither treatment alone was effective. Conclusion The inflammatory response plays a dual role during infection [ 49 , 67 ]. While it is essential for controlling the proliferation and dissemination of pathogens, an excessive or misdirected response can lead to tissue damage and consequent worsening of disease. Our results clearly show that Ang-(1–7) is an important immunomodulatory agent, capable of promoting bacterial clearance and exhibiting enhanced efficacy when associated with antibiotics in a model of pneumococcal sepsis. Thus, this study suggests that the administration of Ang-(1–7) may represent a promising adjunct therapeutic strategy against S. pneumoniae infection in humans. Declarations Authorship Eliza Mathias Melo and Mauro Martins Teixeira wrote the paper and designed the research. Eliza Mathias Melo, Franciel Batista Felix, Izabela Galvão, Flavia Rago, Fernanda Medeiros Vale Magalhães, Marina Gomes Machado, Fernando Roque Ascenção, Geovanni Dantas Cassali performed experiments and analyzes the data. Maria José Campagnole-Santos, Robson Augusto Souza dos Santos, Geovanni Dantas Cassali, Mauro Martins Teixeira provided essential tools and expertise. Funding This investigation received financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) under Grant Agreement No. 476071/2011-9 and no. 309810/2017-5, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil) under Grant Agreement No. PPM-00508-18, The National Institute of Science and Technology in dengue and host-microbial interactions (INCT Dengue CNPq 465425/2014-3), Comissão de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil). Conflict of interest disclosure The authors declare no conflict of interest. Author Contribution EMM and MMT designed the research and wrote the manuscript. EMM, FBF, IG, FR, FMVM, MGM, FRA, GDC performed experiments and analyzes the data. MJCS, RASS, GDC, MMT provided essential tools and expertise. 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15:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6812678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6812678/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00011-025-02146-w","type":"published","date":"2025-11-26T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85140165,"identity":"0825e925-e9f6-4c25-aa29-dd350ef7fb7f","added_by":"auto","created_at":"2025-06-22 09:49:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":351680,"visible":true,"origin":"","legend":"\u003cp\u003eAngiotensin-(1-7) treatment controls inflammatory parameters by reducing leukocyte influx, edema and lung injury after pneumococcal infection. (a) C57BL/6JUnib mice were infected intranasally with 5x10\u003csup\u003e4\u003c/sup\u003e CFU of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e. Treatments with Vehicle (Veh - 92 μg/Kg of HPβCD) or Angiotensin-(1-7) [60 μg/Kg of Ang-(1-7) plus 92 μg/Kg of HPβCD] were every 12 hours by gavage and started 12 to 36 hours after infection. Mice were euthanized 48 hours after infection. (b) Leukocytes, (c) neutrophils and (d) mononuclear cells from BALF were counted based on morphology. (e) MPO activity was measured from mouse lungs. (f) Edema was evaluated by quantifying proteins in BALF by Bradford assay. (g) Histopathological score (maximal of 18) evaluated airway, vascular, parenchymal inflammation, neutrophilic infiltration and their representative images of lung slides were taken at 20x magnification. Groups were presented as median and n = 7, with NI shown as line, and Veh and Ang-(1-7) as individual values. Statistical analysis was done using One-way ANOVA, Turkey’s multiple comparisons test. P values were shown\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/181832b3755cae7404c3b081.png"},{"id":85140085,"identity":"0b07237c-61c5-4485-8379-bc47af78a279","added_by":"auto","created_at":"2025-06-22 09:41:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40748,"visible":true,"origin":"","legend":"\u003cp\u003eAngiotensin-(1-7) administration controls inflammatory parameters by decreasing cytokine and chemokine levels after pneumococcal infection.\u003cstrong\u003e \u003c/strong\u003e(a) Levels of CXCL-1, CCL-2, IL-6, TNF-α, and IL-10 were quantified in BALF by ELISA assay. Groups were presented as median and n = 7, with NI shown as line and Veh and Ang-(1-7) as individual values. Statistical analysis was done using One-way ANOVA, Turkey’s multiple comparisons test. P values were shown\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/87f1ce453c09bb1d2ef2fddb.png"},{"id":85140086,"identity":"45145064-c4ee-457f-b5b4-5a25b7bd7a6f","added_by":"auto","created_at":"2025-06-22 09:41:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27440,"visible":true,"origin":"","legend":"\u003cp\u003eAdministration of Angiotensin-(1-7) improves the survival of mice after Streptococcus pneumoniae infection. (a) C57BL/6JUnib mice were infected intranasally with 5x10\u003csup\u003e4\u003c/sup\u003e CFU of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e. Treatments with Vehicle (Veh - 92 μg/Kg of HPβCD) or Angiotensin-(1-7) [60 μg/Kg of Ang-(1-7) plus 92 μg/Kg of HPβCD] were every 12 hours by gavage and started 12 to 72 hours after infection (b) Mice monitored daily for 10 days to analyze the probability of survival (percent) after infection and n = 9. Statistical survival analysis was done using Log-rank test curve comparison. P values were shown\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/d8d8728b29fab44eba9a225d.png"},{"id":85140087,"identity":"3f2a7ab1-1cd5-4392-9d65-9ec8c0b1cdb1","added_by":"auto","created_at":"2025-06-22 09:41:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15812,"visible":true,"origin":"","legend":"\u003cp\u003eAngiotensin-(1-7) treatment reduces colony forming unit (CFU) in the airways and prevents sepsis by pneumococcal infection. (a) BALF and (b) blood of mice treated from 12 to 36 hours [Veh - 92 μg/Kg of HPβCD and Ang-(1-7) - 60 μg/Kg of Ang-(1-7) plus 92 μg/Kg of HPβCD] were collected for counting of CFU 48 h after \u003cem\u003eS. pneumoniae\u003c/em\u003e infection. Groups were presented as median and n = 6, with NI shown as line, and Veh and Ang-(1-7) as individual values. Statistical analysis was done using unpaired t test. P values were shown\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/c4594b753dada4453548d73c.png"},{"id":85140167,"identity":"ffef9f13-9ed4-4ad7-854f-5b0c77417414","added_by":"auto","created_at":"2025-06-22 09:49:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29120,"visible":true,"origin":"","legend":"\u003cp\u003eAngiotensin-(1-7) treatment to improve phagocytosis of Streptococcus pneumoniae by bone marrow derived macrophages. (a) Phagocytosis of bacteria was evaluated in BMDMs pretreated with Veh (92 μg/Kg of HPβCD) or Ang-(1-7) [60 μg/Kg of Ang-(1-7) plus 92 μg/Kg of HPβCD]. (b) results were expressed as CFU of internalized \u003cem\u003eS. pneumoniae\u003c/em\u003e. Groups were presented as median and n = 7, with NI shown as line and Veh and Ang-(1-7) as individual values. Statistical analysis was done using Mann-Whitney test. P values were shown\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/ababf85d89e53488ec7ad400.png"},{"id":85140090,"identity":"8125f723-bace-4683-85c1-3177d654ed05","added_by":"auto","created_at":"2025-06-22 09:41:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18136,"visible":true,"origin":"","legend":"\u003cp\u003eAngiotensin-(1-7) treatment induces expression of tight junction genes after pneumococcal infection. Relative expression of (a) \u003cem\u003eCldn3\u003c/em\u003e and (b) \u003cem\u003eCldn5\u003c/em\u003e in lung was performed with q-PCR assay. Groups were presented as median and n = 7, with NI shown as line and Veh and Ang-(1-7) as individual values. Statistical analysis was done using One-way ANOVA, Turkey’s multiple comparisons test. P values were shown\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/5669ab26a5a2ea705dc5439f.png"},{"id":85140097,"identity":"4b73d0e1-10c4-49f5-b72d-cba05124b6f1","added_by":"auto","created_at":"2025-06-22 09:41:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":35187,"visible":true,"origin":"","legend":"\u003cp\u003eAntibiotic ceftriaxone and Angiotensin-(1-7) together prevent deaths in case of sepsis due to Streptococcus pneumoniae. (a) C57BL/6JUnib mice were infected intranasally with 5x10\u003csup\u003e5\u003c/sup\u003e CFU of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e. Treatments with Vehicle (Veh - 92 μg/Kg of HPβCD) or Angiotensin-(1-7) [60 μg/Kg of Ang-(1-7) plus 92 μg/Kg of HPβCD] were given by gavage and antibiotic ceftriaxone (ATB 10 mg/Kg) was administered i.p.. All treatments were given 60 and 72 hours after infection. (b) Mice monitored daily for 10 days to analyze the probability of survival (percent) after infection and n = 7. Statistical survival analysis was done using Log-rank test curve comparison. P values were shown between Ang-(1-7) + ATB\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/75e3e86fbf0a9030029e43b6.png"},{"id":97178387,"identity":"fa52b9dc-7df2-4c33-bfaa-d41aed4743c3","added_by":"auto","created_at":"2025-12-01 16:09:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1642118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6812678/v1/a832475a-77a7-45a9-adf4-4b81709a4f53.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Angiotensin-(1-7) treatment improves pneumonia and prevents sepsis caused by pneumococcal infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e is the main cause of community-acquired pneumonia (CAP) irrespective of the severity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Resistance of \u003cem\u003eS. pneumoniae\u003c/em\u003e to antibiotics is increasing in many parts of the world, an observation that makes CAP an infectious disease of significant interest. In addition, CAP represents a substantial clinical and economic burden on society [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among pneumococci, serotype 3 is commonly associated with invasive and lethal infections [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The pathogenesis of this infection includes an important virulence factor, the capsule surrounding \u003cem\u003eS. pneumoniae\u003c/em\u003e, which helps the bacteria escape from phagocytosis and induce intense inflammatory responses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This inflammation triggered by pneumococcal infections is essential for bacterial clearance; however, it must be tightly regulated to preserve alveolar structure, prevent bacterial dissemination from the lungs to the bloodstream, and ultimately avoid fatal outcomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bacterial dissemination is a frequent occurrence in patients with pneumonia, often contributing to extrapulmonary manifestations, including the development of sepsis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe production of pro-resolving molecules during inflammation is crucial to ensure the timely resolution of the immune response and to promote the proper return of the affected tissues to homeostasis after infection [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, pro-resolving molecules may be useful in the context of pulmonary infections by limiting lung inflammation without impairing the host defenses against \u003cem\u003eS. pneumoniae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAngiotensin-(1\u0026ndash;7) [Ang-(1\u0026ndash;7)] is a hormone within the Renin-Angiotensin System (RAS) that recognized for its anti-inflammatory, anti-fibrotic and pro-resolving effect in various acute and chronic models of inflammatory disease [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and, more recently, in infection models [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The effects of Ang-(1\u0026ndash;7) during pneumonia and sepsis caused by \u003cem\u003eS. pneumoniae\u003c/em\u003e has not been evaluated.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design, ethical statement and mice\u003c/h2\u003e \u003cp\u003eThis work was randomized, blinded and controlled by vehicle group during the experimental and analysis stages. All experimental procedures described here were conducted according to Brazilian guidelines on animal work and approved by the local Animal Ethics Committee from Universidade Federal de Minas Gerais (CETEA - UFMG - protocol number 65/2021). C57BL/6JUnib mice aged 8 to 11 weeks were maintained in a controlled environment with free access to filtered water and standard laboratory chow \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStreptococcus pneumoniae\u003c/h3\u003e\n\u003cp\u003eATCC 6303 serotype 3 was grown in culture medium containing on 3.7% Todd Hewitt, 0.5% yeast extract, 5% blood, and 1.5% agar for 12 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Colonies grown on plates were picked and inoculated into Todd-Hewitt broth with 0.5% yeast extract at an initial absorbance (optical density at 600 nm\u0026thinsp;=\u0026thinsp;0.1) and grown to logarithmic phase (optical density at 600 nm\u0026thinsp;=\u0026thinsp;0.4). \u003cem\u003eS. pneumoniae\u003c/em\u003e was then centrifuged at 2,000 rpm for 20 min. Inoculum for infections were prepared after dilution in 0.9% saline as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In all experiments, the inoculum was confirmed by plating of bacterial suspension.\u003c/p\u003e\n\u003ch3\u003eExperimental procedure\u003c/h3\u003e\n\u003cp\u003eAnesthetic solution of xylazine (10 mg/Kg) and ketamine (70 mg/Kg) was administered subcutaneously to the mice. Pneumococcal infection was performed by intranasal instillation with an inoculum of 40 \u0026micro;L of \u003cem\u003eS. pneumoniae\u003c/em\u003e containing 5 x 10\u003csup\u003e4\u003c/sup\u003e or 5 x 10\u003csup\u003e5\u003c/sup\u003e CFU and not infected (NI) mice received 40 \u0026micro;L of sterile saline by intranasal instillation. All mice were monitored until complete recovery from anesthesia. The oral formulation of Ang-(1\u0026ndash;7) was administered via gavage every 12 hours, from 12 to 36 hours following pneumococcal infection. Mice were euthanized 48h after pneumococcal infection to assess the inflammatory response. Broncho alveolar lavage fluid (BALF) was collected to evaluate leukocyte recruitment, cytokine and chemokine production, bacterial loads and protein extravasation. Blood was collected to assess the presence of sepsis and lungs were harvested for histology, myeloperoxidase (MPO) assessment and quantification of tight junction complex expression genes. In lethality experiment, mice weights were followed for 10 days and those who reached 75% of the initial body weight were euthanized. Based on what happens with septic patients, the intraperitoneal (i.p.) ceftriaxone antibiotic (ATB) was associated with Ang-(1\u0026ndash;7) to enhance the effect against sepsis. For oral administration, the pneumococcal infected groups received vehicle (Veh \u0026minus;\u0026thinsp;92 \u0026micro;g/Kg of HPβCD in filtered water, 100 \u0026micro;L, by gavage) and Ang-(1\u0026ndash;7) group received Ang-(1\u0026ndash;7) / HPβCD [60 \u0026micro;g/Kg of Ang-(1\u0026ndash;7) and 92 \u0026micro;g/Kg of HPβCD in filtered water, 100 \u0026micro;l/ mouse, by gavage]. The control non infected group received 100 \u0026micro;l/ mouse of filtered water. The dose used was based on previous studies using murine models of flu [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], asthma [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], arthritis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], emphysema [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and pulmonary fibrosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For intraperitoneal administration, groups infected with \u003cem\u003eS. pneumoniae\u003c/em\u003e, both Veh and Ang-(1\u0026ndash;7), received or not ATB (10 mg/Kg in sterile saline, 100 \u0026micro;L) as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBronchoalveolar lavage fluid (BALF), tissue extraction and total/differential cell count\u003c/h3\u003e\n\u003cp\u003eBALF, tissue extraction and cell count were obtained as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For the BALF, two aliquots of 1mL of PBS were flushed three times into the lungs, through a 1.7mm catheter inserted into the trachea to collect leukocytes and \u003cem\u003eS. pneumoniae\u003c/em\u003e from the alveoli of mice. Thereafter, the left lung was collected and embedded in neutral buffered formalin (10%) for histological analysis and the right lobe was divided for PCR and MPO analyses. Then, BALF was serially diluted and plated in blood agar for bacterial counts (the same was done with blood). Then, BALF samples (2 mL each mouse) were centrifuged at 600 x g for 10 minutes at 4\u003csup\u003eo\u003c/sup\u003eC. Total number of leukocytes was determined by counting leukocytes in a Neubauer chamber. For the differential count, the percentage of each leukocyte (mononuclear and polymorphonuclear) was based on morphological criteria after staining with May\u0026ndash; Gr\u0026uuml;nwald \u0026ndash;Giemsa of slides obtained from cytospin (Shandon III) preparations. Each slide was counted three times, and the percentage was used to calculate the absolute number of each leukocyte type. In addition, BALF supernatants were used for cytokine evaluation by ELISA (R\u0026amp;D Systems, Minneapolis, MN, USA) and total protein quantification using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).\u003c/p\u003e\n\u003ch3\u003eBradford assay\u003c/h3\u003e\n\u003cp\u003ePulmonary edema was assessed by quantifying total protein concentration in BALF. Protein levels were measured using a colorimetric assay, with bovine serum albumin (BSA) as the standard curve ranged from 0.125 to 2 mg/mL. After adding 2 \u0026micro;L of the standard curve and samples to the 96-well plate, 200 \u0026micro;L of Protein Assay Dye Reagent Concentrate (Bio-Rad) diluted 5x was added for detection. The reaction was measured in a spectrophotometer (BioTek Epoch \u0026ndash; Agilent Technologies) at an absorbance read at 590 nm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISA assay\u003c/h2\u003e \u003cp\u003eConcentrations of TNF-α, IL-6, CCL-2 and CXCL-1 were measured in the BALF supernatants by ELISA DuoSet kits according to the procedures supplied by the manufacturer (R\u0026amp;D Systems, Minneapolis, MN, USA). Briefly, NuncFlat 96-well plates were sensitized with specific detection antibodies diluted in PBS and incubated overnight. Then the plates were washed with 0.1% (v/v) Tween, blocked with 1% (v/v) bovine serum albumin (BSA) for 2 h and washed again after blocking. 100 \u0026micro;L of samples, standards and blank were added to the plates and incubated overnight. The next day, the plates were washed again and detection antibodies diluted in PBS were added. Another round of washing was performed, and streptavidin solution was added to the wells. OPD and oxygen peroxide were added to the plates for 20 min to generate a colorimetric reaction. The reaction was measured in a spectrophotometer (BioTek Epoch \u0026ndash; Agilent Technologies) at 490 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMyeloperoxidase assay - MPO\u003c/h3\u003e\n\u003cp\u003eThe right lung of mice was collected for indirect quantification of neutrophils recruitment into the tissue (MPO \u0026ndash; myeloperoxidase assay). As previously described [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], 50 mg of lung tissue were homogenized in a buffered solution containing antiproteases. MPO levels were performed using 25 \u0026micro;L of the supernatant of the homogenized and 25 \u0026micro;L of a solution of 3,39\u0026thinsp;\u0026minus;\u0026thinsp;5,59-tetramethylbenzidine (1.6 mM, TMB; Sigma \u0026ndash; dissolved in dimethyl sulfoxide) and 100 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.01 mM) diluted in phosphate buffer (pH 5.4) containing HETAB.\u003c/p\u003e\n\u003ch3\u003eBone Marrow-Derived Macrophages (BMDMs) isolation\u003c/h3\u003e\n\u003cp\u003eBMDM were obtained as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, following euthanasia, tibias and femurs were collected, and bone marrow cells were flushed out using sterile medium for subsequent differentiation into macrophages. The cell suspension obtained was then centrifuged at 1200 RPM for 5 min. The pellet was resuspended in complete conditioned media for BMDM differentiation (RPMI containing 10% heat-inactivated fetal bovine serum and 30% L929 cell-conditioned medium), seeded on petri dishes and incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. After 7 days, the supernatant was discarded, and adherent macrophages were detached by using a cell scraper and plated (2 x 10\u003csup\u003e5\u003c/sup\u003e cells/well) in 96-well plates for phagocytosis assay.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhagocytosis assay\u003c/h2\u003e \u003cp\u003ePhagocytosis was evaluated as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, 2 x 10\u003csup\u003e5\u003c/sup\u003e BMDMs were plated and incubated for 3 h for adhesion at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Then, treatments Ang-(1\u0026ndash;7) (152 \u0026micro;g/Kg of Ang-(1\u0026ndash;7) / HPβCD) or Veh (92 \u0026micro;g/Kg of HPβCD) was added to the well for 18 h and then a MOI of 10 of \u003cem\u003eS. pneumoniae\u003c/em\u003e was added to this culture of BMDMs for 3 h to allow phagocytosis (1 h of adhesion at 4\u0026deg;C followed by 2 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e). Noninternalized bacteria were washed out with 30 \u0026micro;g/mL of penicillin and streptomycin for 30 min. Then, BMDMs were briefly lysed with cold water and then plated on blood agar for subsequent CFU counts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene expression\u003c/h2\u003e \u003cp\u003eGene expression was obtained as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 50 mg of the right lung lobe was collected and stored at -80\u0026deg;C until use. Total RNA was isolated from this tissue incubated and minced in 500 \u0026micro;L TRIzol (Invitrogen). Then, 200 \u0026micro;L of chloroform was added, and the samples were vortexed for 15 s before being centrifuged at 12,000 g for 15 min at 4\u0026deg;C. Aqueous phase samples were collected and 500 \u0026micro;L isopropanol was added to them. Samples were incubated on ice for 10 min and then were centrifuged at 12,000 g for 15 min at 4\u0026deg;C. The supernatants were discarded, and the pellet was washed with 75% ethanol solution. The pellet was dried at room temperature and then resuspended with 20 \u0026micro;L of injection water. RNA concentration was measured by nanodrop and then stored at -80\u0026deg;C. For cDNA preparation, samples were treated with DNAse and RNA template was converted to cDNA by reverse transcriptase using Superscript Transcriptase (Promega), according to the manufacturer\u0026rsquo;s instructions. cDNA was stored at -20\u0026deg;C until quantitative PCR (q-PCR) assays. qPCR was performed Sybr Green PCR Master Mix (Applied Biosystems) with addition of forward and reverse primers and samples. Runs were performed on the 7500 Fast System. Tight junction primers were designed to amplify exon-exon junctions in mRNA and the relative level of gene expression was determined by the 2^(-ΔΔ Ct) method. Data for each sample were normalized to GAPDH mRNA and expressed as relative expression. The primers used were epithelial barrier genes m-Cldn3 FWD 5\u0026prime;-CCAACTGCGTACAAGACGAG-3\u0026prime;; m-Cldn3 REV 5\u0026prime;-TCTTGGTGGGTGCATACTTG-3\u0026prime;; m-Cldn5 FWD 5\u0026prime;-TGGAACGCTCAGATTTCATC-3\u0026prime; and m-Cldn5 REV AGGAAGGCAACCCCTCTAAG-3\u0026prime;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eAfter the mice's left lungs were fixed in formalin solution, they were gradually dehydrated in ethanol and processed as described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 4 \u0026micro;m sections of the tissue were stained with Hematoxylin \u0026amp; Eosin and analyzed by a pathologist blinded to the experiment. The score of inflammation totaled 18 points and were distributed when evaluating airway inflammation (up to 4 points), vascular inflammation (up to 4 points), parenchymal inflammation (up to 5 points) and general neutrophil infiltration in the lungs (up to 5 points).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistics were performed using GraphPad Prism 9.0. Before comparing groups, the Shapiro-Wilk normality test was performed. Then, one-way ANOVA followed by Turkey\u0026rsquo;s multiple comparisons test was used to compare more than two groups, and the unpaired t-test was used for comparisons between two groups. Survival curves were analyzed by the Long-rank test. Results with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eAdministration of Angiotensin-(1\u0026ndash;7) reduced leukocyte infiltration, improved edema formation and lung damage after pneumococcal infection\u003c/h2\u003e\n \u003cp\u003eThe first experiments evaluated the effects of Ang-(1\u0026ndash;7) treatment in pneumonia caused by \u003cem\u003eS. pneumoniae\u003c/em\u003e. Mice were infected intranasally with 5x10\u003csup\u003e4\u003c/sup\u003e CFU and then treated with Ang-(1\u0026ndash;7) or Veh from 12 to 36 h after pneumococcal infection. Animals were euthanized 48 h after infection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Ang-(1\u0026ndash;7) treatment reduced the total number of leukocyte and neutrophils in BALF at 48 h, as compared to Veh-treated animals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). There was no difference in mononuclear cell recruitment between mice treated with Ang-(1\u0026ndash;7) and Veh in the airways after pneumococcal infection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). In addition, MPO activity in the lungs of mice treated with Ang-(1\u0026ndash;7) tended to decrease when compared to Veh (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). Pulmonary edema, measured by the concentration of proteins that leaked into the airways, was lower in mice treated with Ang-(1\u0026ndash;7) than Veh (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef). Consistent with the control of neutrophil and leukocyte recruitment and reduction in pulmonary edema, evaluation of lung tissue injury in histopathological sections show that damage was lower in mice treated with Ang-(1\u0026ndash;7) than Veh after pneumococcal infection (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). These results suggest reduced inflammatory parameters when mice are treated with Ang-(1\u0026ndash;7) after pneumococcal infection.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eLevels of pro-inflammatory cytokines and chemokines are decreased in the airways of Angiotensin-(1\u0026ndash;7)-treated mice after pneumococcal infection\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003ePro-inflammatory cytokines and chemokines that remain persistent and contribute to robust acute inflammation are also associated with tissue damage, morbidity and, consequently, death. Thus, Ang-(1\u0026ndash;7) treatment, compared to Veh, has been shown to reduce CXCL-1 levels (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), consistent with the lower neutrophil influx observed. Furthermore, compared to Veh, Ang-(1\u0026ndash;7) treatment reduced CCL-2 and IL-6 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, c, respectively), which are important in the control of mononuclear cells and acute inflammation. In addition, Ang-(1\u0026ndash;7) treatment reduced TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed) associated with the lower tissue damage demonstrated. IL-10, which has an anti-inflammatory role, remained the same between the different groups analyzed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). Overall, we showed that Ang-(1\u0026ndash;7) treatment was able to reduce the levels of pro-inflammatory mediators, contributing to a finely regulated acute inflammation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAdministration of Angiotensin-(1\u0026ndash;7) improved the survival of mice after Streptococcus pneumoniae infection\u003c/h2\u003e\n \u003cp\u003eTo evaluate the effects of Ang-(1\u0026ndash;7) treatment on the lethality rates, we infected mice with \u003cem\u003eS. pneumoniae\u003c/em\u003e, treated them with Ang-(1\u0026ndash;7) or Veh every 12 hours from 12 to 36 hours after infection. Mice were monitored the mice daily for 10 days to assess the survival rate (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). We were able to observe that Ang-(1\u0026ndash;7) treatment increased survival when compared to Veh after pneumococcal infection (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). These data confirmed that controlling inflammatory parameters and tissue damage results in reduction of deaths caused by \u003cem\u003eS. pneumoniae\u003c/em\u003e infection.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAngiotensin-(1\u0026ndash;7) treatment reduced colony forming unit (CFU) in the airways and prevented sepsis by pneumococcal infection\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eA finely regulated immune response may contribute to more effective bacterial clearance [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. When comparing Veh and Ang-(1\u0026ndash;7) treatments, we observed that Ang-(1\u0026ndash;7) not only prevented mortality but also finely modulated the immune response. We were also able to observe that this was associated with better control of bacteria replication, as seen by the lower number of \u003cem\u003eS. pneumoniae\u003c/em\u003e in the airways and blood (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, respectively). Therefore, although lung inflammation was greatly reduced in mice treated with Ang-(1\u0026ndash;7), the level of inflammation observed was sufficient to control \u003cem\u003eS. pneumoniae\u003c/em\u003e numbers in the alveoli and prevent dissemination to blood.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eAngiotensin-(1\u0026ndash;7) treatment acted on bone marrow derived macrophages (BMDMs) to improve phagocytosis of Streptococcus pneumoniae\u003c/h2\u003e\n \u003cp\u003eThe lower amount of \u003cem\u003eS. pneumoniae\u003c/em\u003e in the airways and blood may be due to the improved ability of the cells to deal with this pathogen or may be due to a direct antibacterial action of the molecule. First, we evaluated the effect of treatment of Ang-(1\u0026ndash;7) in BMDMs infected with \u003cem\u003eS. pneumoniae\u003c/em\u003e. To this end, BMDMs were treated with Veh or Ang-(1\u0026ndash;7) for 18 h, infected with a MOI of 10 of \u003cem\u003eS. pneumoniae\u003c/em\u003e for 2 h and then plated for a further 3 h (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Treatment with Ang-(1\u0026ndash;7) clearly increased the phagocytosis of \u003cem\u003eS. pneumoniae\u003c/em\u003e by BMDMs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, there was no direct effect of Ang-(1\u0026ndash;7) on \u003cem\u003eS. pneumoniae in vitro\u003c/em\u003e, as the bacterium maintained its usual growth phases in the groups treated with Veh or Ang-(1\u0026ndash;7) (Supplementary Fig. 1). Thus, Ang-(1\u0026ndash;7) acts on BMDMs and improves their phagocytic capacity, but has no direct bactericidal, bacteriostatic or even bacteriolytic action.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAdministration of Angiotensin-(1\u0026ndash;7) promoted increased expression of tight junction genes in lung tissue after pneumococcal infection\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDuring bacterial infections, uncontrolled bacterial replication can cause loss of the epithelial barrier, allowing the pathogen to leak into the bloodstream and cause sepsis [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. To evaluate the epithelial barrier, we assessed the effects of treatment with Ang-(1\u0026ndash;7) on the expression of genes (\u003cem\u003eCldn3\u003c/em\u003e and \u003cem\u003eCldn5\u003c/em\u003e) associated with the tight junction complex. Treatment with Ang-(1\u0026ndash;7) was associated with an increase in the expression of \u003cem\u003eCldn3\u003c/em\u003e and \u003cem\u003eCldn5\u003c/em\u003e in the lungs (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, respectively), suggesting lower \u003cem\u003eS. pneumoniae\u003c/em\u003e dissemination may have been secondary to greater epithelial barrier integrity.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCombining the antibiotic ceftriaxone and Angiotensin-(1\u0026ndash;7) as treatments in cases of severe pneumonia caused by Streptococcus pneumoniae resulted in even greater survival\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn cases of severe pneumonia, sepsis may occur and is associated with a significant increase in mortality. Providing adequate antibiotic treatment is crucial but is often started too late in the course of disease at a time it is no possible to reverse the septic condition [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. In attempt to model this situation in our animal model of infection, mice were infected intranasally with a higher inoculum of \u003cem\u003eS. pneumoniae\u003c/em\u003e (5x10\u003csup\u003e5\u003c/sup\u003e CFU) and then treated with Ang-(1\u0026ndash;7) in the absence or presence of and antibiotic (ceftriaxone) or Veh from 60 to 72 h after infection (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea). Using this inoculum, 100% of animals were dead by day 6 after infection. Using this higher inoculum and a more delayed initiation of treatment, Ang-(1\u0026ndash;7) alone was not associated with enhanced survival. Similarly, delayed start of Ceftriaxone treatment alone was not associated with significantly enhanced survival (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). In contrast, delayed start of treatment with a combination of Ang-(1\u0026ndash;7) and ceftriaxone greatly prevented death in this model of severe and disseminated pneumococcal infection.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePneumonia caused by \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e remains a significant global health threat. According to the World Health Organization (WHO), in 2008, it was estimated that 541,000 children under the age of five died from pneumococcal diseases, with pneumonia being the primary manifestation. Mortality rates from pneumococcal infections can be as high as 20% in cases of severe pneumonia accompanied by sepsis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The severity of pneumococcal pneumonia is largely driven by the inflammatory response it triggers. Exaggerated inflammation, if not properly regulated, can lead to excessive tissue damage and worsen the infection. In our study, we demonstrated that (i) exogenous administration of Ang-(1\u0026ndash;7) significantly reduced leukocyte infiltration, alleviated pulmonary edema, decreased TNF-α levels, while treatment enhanced bacterial clearance and improved both survival rates. Importantly, (ii) in a model of severe and invasive pneumococcal infection, a critical and often fatal complication in hospitalized patients, Ang-(1\u0026ndash;7) exhibited a synergistic effect when combined with antibiotics, resulting in increased survival. Mechanistically, (iii) treatment with Ang-(1\u0026ndash;7) enhanced phagocytosis of bacteria, an effect associated with decreased bacterial loads in the lung, and enhanced epithelial cell integrity, an effect associated with decreased bacterial loads in blood.\u003c/p\u003e \u003cp\u003eAng-(1\u0026ndash;7) is an endogenous heptapeptide part of the counter-regulatory branch of the Renin-Angiotensin System (RAS) and whose bioactivity extends far beyond the cardiovascular system [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Our studies have clearly demonstrated that Ang-(1\u0026ndash;7) has anti-inflammatory and pro-resolving effects \u003cem\u003ein vivo\u003c/em\u003e It has been reported to reduce lung inflammation, fibrosis, pulmonary arterial hypertension and prevent secondary pneumococcus infection after influenza [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings that administration of Ang-(1\u0026ndash;7) makes the course of pneumococcal disease less severe, as seen by the reduction of leukocyte infiltration, mainly neutrophils in the pulmonary alveoli are consistent with the anti-inflammatory and pro-resolving actions of Ang-(1\u0026ndash;7) in several systems [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. They are also consistent with the effects of other pro-resolving molecules in models of pneumonia in which protection was associated with decreased neutrophil recruitment and activation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In contrast, the course of the disease was worse in mice that had their neutrophils depleted [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Therefore, it is clear that a certain level of neutrophil recruitment and activation is necessary in the context of pneumococcal and other bacterial infections. However, excessive neutrophil influx and activation may be detrimental to the host and lead to pulmonary injury and death [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe were also able to observe less pulmonary edema and lower amounts of TNF-α, which are closely linked to more intact lung tissue, demonstrating less neutrophil infiltration, less inflammation of the parenchyma, vascular and airways after therapeutic administration of Ang-(1\u0026ndash;7). Interestingly, absence of pro-resolution agents leads to an intense increase in TNF-α, causing endotoxic shock in a model of challenge with LPS [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This exacerbated increase in TNF-α also contributes to the pathogenesis and development of pulmonary edema [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and causes multiple inflammatory disorders [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Pulmonary edema is a significant medical problem worldwide and can be life-threatening because it is related to important clinical manifestations such as shock, diffuse alveolar damage and lung hypersensitivity states [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, controlling TNF-α levels, reducing histopathological damage and pulmonary edema contribute to better outcomes related to pneumonia and sepsis due to \u003cem\u003eS. pneumoniae\u003c/em\u003e. Not only TNF-α, but other cytokines, such as IL-10 and IL-6, are related to severe clinical signs of pneumonia caused by \u003cem\u003eS. pneumoniae\u003c/em\u003e [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and predict severe progression of the disease [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In addition, these levels are reduced after antibiotic treatment [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In the same way, it was possible to see a reduction in IL-6 and TNF-α, after the administration of Ang-(1\u0026ndash;7) treatment.\u003c/p\u003e \u003cp\u003eWith these various inflammatory parameters limited due to Ang-(1\u0026ndash;7) administration, there was consequently greater survival after pneumococcal infection.\u003c/p\u003e \u003cp\u003eReduction of severity of pneumonia is associated with reduced pulmonary injury, reduced bacterial dissemination (sepsis), and fewer early deaths [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. We have previously shown that Ang-(1\u0026ndash;7) was also highly protective against severe primary Influenza A virus (IAV) infection and protected against secondary \u003cem\u003eS. pneumoniae\u003c/em\u003e infection in the lung leading [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Other pro-resolving molecules have been shown to be protective in models of pulmonary infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. For example, the pro-resolving molecule Annexin A1 (AnxA1) also prevented excessive lung damage and the absence of AnxA1 increased lethality after pneumococcal infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Pro-resolving molecules are also effective in systemic sepsis from a source other than the lung. Indeed, administration of pro-resolving plasminogen/plasmin (Plg/Pla) protected mice from sepsis-induced by cecal ligation and puncture (CLP) lethality and enhanced the protective effect of antibiotics [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Reduced mortality was clearly associated with decreased pulmonary inflammation, edema, and tissue damage, as assessed histologically. Improved survival was also linked with greater control of bacterial replication in the lungs and reduced dissemination into the bloodstream, indicating decreased progression to sepsis.\u003c/p\u003e \u003cp\u003eA reduction in bacterial load in both the lungs and bloodstream may have clearly contributed to the decreased severity of pneumococcal pneumonia and sepsis. It is important to note that Ang-(1\u0026ndash;7) had no direct antimicrobial impact on \u003cem\u003eS. pneumoniae\u003c/em\u003e. However, Ang-(1\u0026ndash;7) greatly enhanced the ability of macrophages to phagocytose \u003cem\u003eS. pneumoniae\u003c/em\u003e. Indeed, we have previously shown that Ang-(1\u0026ndash;7) acts as a phagocyte attractant and increased the ability of BMDMs to phagocytose \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In experimental Type 2 Diabetes Mellitus, Ang-(1\u0026ndash;7) rescued the ability of mice neutrophils to phagocytose \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, a bacterium that also causes lung infection [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Additionally, in a model of \u003cem\u003eMycoplasma pneumonia\u003c/em\u003e infection, Ang-(1\u0026ndash;7) demonstrated efficacy in decreasing colonization of this pathogen, known to provoke exacerbations in asthma and chronic obstructive pulmonary disease (COPD) patients, in the airways [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, the unique capacity of Ang-(1\u0026ndash;7) to enhance phagocytosis of bacteria by macrophages was associated with decreased number of bacteria in the lungs and these effects may have contributed to the ability of Ang-(1\u0026ndash;7) to decreased severity and deaths in this model of infection.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. pneumoniae\u003c/em\u003e may replicate in the lungs and disseminate to other tissues, causing sepsis and high degree of lethality [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In this regard, not only the function of leukocytes undergoing phagocytosis, but also other factors such as lung epithelial barrier integrity may be important to prevent the occurrence of dissemination and sepsis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Greater integrity of the epithelial barrier may also contribute to lower lung injury and edema contributing to lower fatalities after infection [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Our results show that the gene expression of claudins 3 and 5, which are part of the tight junction complex (TJC) and hence relevant for epithelial barrier integrity, was increased after Ang-(1\u0026ndash;7) administration. The reduction in IL-6 may have contributed to the increased expression of \u003cem\u003eCldn3\u003c/em\u003e and \u003cem\u003eCldn\u003c/em\u003e5 in the lung after Ang-(1\u0026ndash;7) treatment, since there is an inverse correlation of claudins expression and IL-6 production [\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In another study, the absence of pro-resolving AnxA1 impaired the upregulation of \u003cem\u003eCldn3\u003c/em\u003e and \u003cem\u003eCldn5\u003c/em\u003e genes, which might have accounted for the loss of lung barrier integrity during pneumococcal infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the upregulation of these tight junction-associated genes may have contributed to the beneficial effects of Ang-(1\u0026ndash;7) by limiting edema formation and bacterial dissemination into the bloodstream.\u003c/p\u003e \u003cp\u003eFinally, our results showed that delayed administration of Ang-(1\u0026ndash;7) was able to rescue the ability of antibiotics to preventing death after severe pulmonary pneumococcal infection. This is indeed very clinically relevant as both Ang-(1\u0026ndash;7) and the antibiotic were given late in the course of infection when neither treatment alone was effective.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe inflammatory response plays a dual role during infection [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. While it is essential for controlling the proliferation and dissemination of pathogens, an excessive or misdirected response can lead to tissue damage and consequent worsening of disease. Our results clearly show that Ang-(1\u0026ndash;7) is an important immunomodulatory agent, capable of promoting bacterial clearance and exhibiting enhanced efficacy when associated with antibiotics in a model of pneumococcal sepsis. Thus, this study suggests that the administration of Ang-(1\u0026ndash;7) may represent a promising adjunct therapeutic strategy against \u003cem\u003eS. pneumoniae\u003c/em\u003e infection in humans.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthorship\u003c/h2\u003e\n\u003cp\u003eEliza Mathias Melo and Mauro Martins Teixeira wrote the paper and designed the research. Eliza Mathias Melo, Franciel Batista Felix, Izabela Galv\u0026atilde;o, Flavia Rago,\u0026nbsp;Fernanda Medeiros Vale Magalh\u0026atilde;es,\u0026nbsp;Marina Gomes Machado, Fernando Roque Ascen\u0026ccedil;\u0026atilde;o, Geovanni Dantas Cassali performed experiments and analyzes the data. Maria Jos\u0026eacute; Campagnole-Santos, Robson Augusto Souza dos Santos, Geovanni Dantas Cassali, Mauro Martins Teixeira provided essential tools and expertise.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis investigation received financial support from Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq, Brazil) under Grant Agreement No. 476071/2011-9 and no.\u0026nbsp;309810/2017-5, Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil) under Grant Agreement No. PPM-00508-18, The National Institute of Science and Technology in dengue and host-microbial interactions (INCT Dengue CNPq 465425/2014-3), Comiss\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de Ensino Superior (CAPES, Brazil).\u003c/p\u003e\n\u003ch2\u003eConflict of interest disclosure\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eEMM and MMT designed the research and wrote the manuscript. EMM, FBF, IG, FR, FMVM, MGM, FRA, GDC performed experiments and analyzes the data. MJCS, RASS, GDC, MMT provided essential tools and expertise.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSteel HC, Cockeran R, Anderson R, Feldman C. Overview of community-acquired pneumonia and the role of inflammatory mechanisms in the immunopathogenesis of severe pneumococcal disease. Mediators Inflamm. 2013;2013:490346. doi:10.1155/2013/490346\u003c/li\u003e\n\u003cli\u003eWelte T. Risk factors and severity scores in hospitalized patients with community-acquired pneumonia: prediction of severity and mortality. Eur J Clin Microbiol Infect Dis. 2012;31(1):33-47. doi:10.1007/s10096-011-1272-4\u003c/li\u003e\n\u003cli\u003eFile TM Jr, Ramirez JA. Community-Acquired Pneumonia. N Engl J Med. 2023;389(7):632-641. doi:10.1056/NEJMcp2303286\u003c/li\u003e\n\u003cli\u003eFeldman C. 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Clin Microbiol Rev. 2019;32(3):e00107-18. doi:10.1128/CMR.00107-18\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Figure","content":"\u003cp\u003eSupplementary Figure 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Streptococcus pneumoniae, Angiotensin-(1–7), inflammation, sepsis, phagocytosis, infection","lastPublishedDoi":"10.21203/rs.3.rs-6812678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6812678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePneumonia caused by \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e remains a major health issue with significant morbidity and mortality. This study investigates the therapeutic potential of Angiotensin-(1\u0026ndash;7) [Ang-(1\u0026ndash;7)], a peptide with anti-inflammatory and pro-resolving effects, in a murine model of pneumococcal pneumonia. In mice infected with \u003cem\u003eS. pneumoniae\u003c/em\u003e, Ang-(1\u0026ndash;7) reduced leukocyte infiltration, pulmonary edema, and tissue damage, and lowed production of the pro-inflammatory cytokines TNF-α, IL-6, and CXCL-1. The treatment improved bacterial clearance in the bronchoalveolar lavage and blood and increased survival rates. Importantly, when combined with the antibiotic ceftriaxone, Ang-(1\u0026ndash;7) enhanced survival even when treatment started late in this model of pneumococcal pneumonia. Mechanistically, Ang-(1\u0026ndash;7) enhanced the phagocytic activity of \u003cem\u003eS. pneumoniae\u003c/em\u003e by bone marrow-derived macrophages and increased the expression of genes associated with lung barrier integrity. These results show that treatment with Ang-(1\u0026ndash;7) decreases inflammation and improves outcomes in severe and invasive pneumococcal pneumonia, especially when combined with antibiotics, suggesting it may be a useful adjuvant therapeutic strategy in this infectious condition.\u003c/p\u003e","manuscriptTitle":"Angiotensin-(1-7) treatment improves pneumonia and prevents sepsis caused by pneumococcal infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-22 09:41:42","doi":"10.21203/rs.3.rs-6812678/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-06T11:30:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-23T16:24:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164317907608556814367474076665630278367","date":"2025-07-08T17:16:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T14:22:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-05T06:54:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-05T06:51:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation Research","date":"2025-06-03T14:58:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inre","sideBox":"Learn more about [Inflammation Research](http://link.springer.com/journal/11)","snPcode":"11","submissionUrl":"https://submission.nature.com/new-submission/11/3","title":"Inflammation Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f81e1081-5f69-42f4-9c98-59e38663d863","owner":[],"postedDate":"June 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:02:18+00:00","versionOfRecord":{"articleIdentity":"rs-6812678","link":"https://doi.org/10.1007/s00011-025-02146-w","journal":{"identity":"inflammation-research","isVorOnly":false,"title":"Inflammation Research"},"publishedOn":"2025-11-26 15:57:51","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-06-22 09:41:42","video":"","vorDoi":"10.1007/s00011-025-02146-w","vorDoiUrl":"https://doi.org/10.1007/s00011-025-02146-w","workflowStages":[]},"version":"v1","identity":"rs-6812678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6812678","identity":"rs-6812678","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}