Zymomonas mobilis culture protects against sepsis by modulating the inflammatory response, alleviating bacterial burden and suppressing splenocyte apoptosis

European Journal of Pharmaceutical Sciences 48 (2013) 1–8

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Zymomonas mobilis culture protects against sepsis by modulating the inflammatory response, alleviating bacterial burden and suppressing splenocyte apoptosis Ingrid Araújo Campos a, Eulália Azevedo Ximenes a, Carlson Helder R. Carvalho Júnior a, Amanda Rafaela C. de Mesquita a, José Bruno N.F. Silva a, Maria Bernadete S. Maia b, Eryvelton Souza Franco b, Paloma Lys Medeiros c, Christina A. Peixoto d,e, Teresinha Gonçalves da Silva a,⇑ a

Department of Antibiotics, Laboratory of Bioassays for Research on Drugs, Federal University of Pernambuco, Brazil Department of Physiology and Pharmacology, Laboratory of Pharmacology of Bioactive Products, Federal University of Pernambuco, Brazil c Department of Histology and Embryology, Federal University of Pernambuco, Brazil d Laboratory of Ultrastructure, Research Center Ageu Magalhães, Pernambuco, Brazil e Center for Strategic Technology in the Northeast (CETENE), Pernambuco, Brazil b

a r t i c l e

i n f o

Article history: Received 18 April 2012 Received in revised form 1 October 2012 Accepted 2 October 2012 Available online 2 November 2012 Keywords: Sepsis Zymomonas mobilis Apoptosis Cytokines

a b s t r a c t Microorganisms with immunomodulating effects beneficially affect the host organism by improving the microbial equilibrium and balancing the immune system. Zymomonas mobilis is reported to have antagonistic properties against yeast and other pathogenic microorganisms in humans and animals. This study assessed the effects of Z. mobilis UFPEDA 202 (109 CFU/mL) cultures on polymicrobial sepsis induced by cecal ligation and puncture (CLP). The survival of animals subjected to lethal sepsis was evaluated after pre-treatment, post-treatment or a combination of both. 6 h after the induction of sepsis, neutrophil migration, the number of bacteria, myeloperoxidase, TNF-a, MCP-1, and IL-10 were performed in the peritoneal lavage of animals. Histopathological changes in the spleen of animals were evaluated by light microscopy, and apoptosis of splenocytes was analyzed by transmission electron microscopy. The results showed that the combination of prophylactic and therapeutic treatment with Z. mobilis increased the survival of animals by 50% at 96 h after the induction of sepsis. There was a reduction in the levels of TNF-a and myeloperoxidase (MPO) in lung tissue. There was also a reduction in the number of viable bacteria in peritoneal fluid. However, increases in neutrophil migration and IL-10 levels were observed. The observed levels of MCP-1 remained similar to the control. Histopathology analysis showed a decrease in acute lung injury. The group pre-treated with the Z. mobilis culture demonstrated a marked decrease in the number of apoptotic cells in the spleen (24%). This study demonstrates that Z. mobilis cultures increased the survival of animals with severe sepsis. This survival was mediated by improvement of neutrophil migration, enhanced activity against pathogenic enteric bacteria and reduced lung injury. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction The intestinal mucosa may represent a site early in infection and may also be a gateway for pathogenic microorganisms. Thus, the host develops a series of defense mechanisms that are able to eliminate pathogenic microorganisms, including the innate and adaptive immune responses (Vaughan et al., 1999). Certain nonpathogenic microorganisms are capable of stimulating the immune response of host intestinal cells. This interaction occurs with the

⇑ Corresponding author. Address: Departamento de Antibióticos, Universidade Federal de Pernambuco, Rua Prof. Artur Sá, 1235, Cidade Universitária, Recife/PE, Brazil. E-mail address: [email protected] (T.G. da Silva). 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.10.011

lymphoid tissue associated with the gastrointestinal tract (GALT), which begins to form before birth (Eberl and Lochner, 2009). Zymomonas mobilis is a Gram-negative, facultative anaerobic bacterium that ferments glucose, fructose, and sucrose as carbon sources (Doelle et al., 1993). Earlier literature reports by Gonçalves de Lima et al. (1970) have suggested a role of the Z. mobilis as a therapeutic agent. They described the ability of Z. mobilis to antagonize the growth of a wide variety of bacteria and fungi (Gonçalves De Lima et al., 1968). It has also been studied as a probiotic bacterium (Azerêdo et al., 2010; Mesquita, 2008), as antitumor (via production of secondary metabolites) (Calazans et al., 2000), and protective against Schistossoma mansoni (Santos et al., 2004). Z. mobilis strains occur naturally in various fermentations from different carbohydrates as wine of apple and pear in Europe; fermented sap of Agave in Mexico; beer deteriorated in the British

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Isles; sap of several palms in Africa, Asia and the Americas; and fermented sugarcane juice in Brazil. In such fermentations, Z. mobilis are associated with other micro-organisms such as yeast, Lactobacillus, Acetobacter, Leuconostoc, as part of a microflora ‘‘suis generis’’, dependent on the carbon source, sometimes become difficult the isolation (Falcão de Morais, 1982; Falcão de Morais et al., 1983). Probiotics are nonpathogenic microorganisms, which confer benefits to the host when administrated in sufficient amounts (Cong et al., 2003). Previous studies have shown that oral administration of probiotics modulates intestinal immunity, improves the balance of the gut microflora, enhances the recovery of the disturbed gut mucosal barrier, and prevents microbial translocation (Cong et al., 2003; Isolauri et al., 2002). The use of Z. mobilis lies in its potential ability to replace pathogenic microorganisms by commensal bacteria, thereby altering their interaction with the immune system (Foligne et al., 2007). The translocation of pathogenic microorganisms of the gastrointestinal tract to the rest of body can cause sepsis and the destruction of the function of the intestinal barrier. Therefore, the use of the Z. mobilis, with its probable immunomodulatory actions, is justifiable. A breakdown in intestinal barrier function and increased bacterial translocation are key events in the pathogenesis of sepsis. An alteration of the gut microbiota with non-pathogenic microorganisms, as well as the use of immunomodulators, has been proposed as adjuvant therapy to reduce the levels of bacterial translocation and prevent the onset of sepsis (Ewaschuk et al., 2007). Therefore, the aim of this study was to determine the effectiveness Z. mobilis culture in preventing or attenuating the development of sepsis in animal models, likely through its actions on the immune system and decreased translocation of pathogenic bacteria. 2. Materials and methods 2.1. Microorganisms Z. mobilis (UFPEDA-202) was kindly provided by the Collection of Microorganisms, Department of Antibiotics, Federal University of Pernambuco (Recife, Brazil). The strain was cultured in the Standard Swings and De Ley – SSDL broth (glucose 20.0 g L 1, yeast extract 15 g L 1). The number of viable cells (CFU/mL) was determined by the pour plate method using SSDL agar (glucose 20.0 g L 1, yeast extract 15 g L 1, agar 15 g L 1) incubated at 32 °C for 48 h. 2.2. Experimental animals To perform the sepsis experiments induced by cecal ligation and puncture (CLP), we used male mice (Mus musculus) provided by the animal facilities of the Antibiotics Department (UFPE Recife, Brazil). The mice used weighed between 25 and 30 g and were kept in a room with controlled temperature (22 ± 2 °C) and humidity (50–60%) and a 12-h light/dark cycle. Water and food were made available to the animals without restriction. Before beginning the experiments, the animals were acclimated to the laboratory environment for at least 30 min. The Animal Studies Committee of the Federal University of Pernambuco approved the experimental protocols (number 23076.015663/2011-10). The experimental protocols conducted followed the technical and ethical principles recommended by the norms of the National Institute of Health Guide for Care and Use of Laboratory Animals.

to some authors the dose of probiotics acceptable for human consumption is among 109–1011 CFU/day (Gratz et al., 2010). The suspension was given orally (two times a day), with a groupdependent variable duration. In groups with 16 animals, half were euthanized after 6 h to assess specific parameters (leukocytes count, determination of cytokines, myeloperoxidase, bacteria counts in peritoneal fluid, histopathological evaluation and apoptosis of splenocytes). The other half of each group was used for evaluation of the survival rate. A total of 64 animals were divided into five groups. The C-CLP control group (1) contained animals (n = 16) that received saline solution (0.9%) for 10 days. On the 11th day, lethal sepsis was induced by CLP. Half of the animals were euthanized, while the other half was used for evaluation of the survival rate. The PT-CLP group (2) received prophylactic treatment with Z. mobilis, in which animals (n = 16) were treated for 10 days before the induction of sepsis. On the 11th day, lethal sepsis was induced by CLP. Half of the animals were euthanized after 6 h, while the other half was used for evaluation of the survival rate. In the Sham group (3), animals (n = 16) received a saline solution (0.9%) for 10 days. On the 11th day, half of the animals underwent an incision of 2 cm in the abdomen without bowel manipulation or induction of sepsis. The animals were then sutured and euthanized 6 h after the procedure. The other half of the group was used for evaluation of the survival rate. In the T-CLP group (4) (the therapeutic group) CLP-induced animals (n = 8) were treated with Z. mobilis two times a day for 96 h and used for the evaluation of the survival rate. In the PPT-CLP group (5) (the prophylactic and therapeutic group), the animals (n = 8) were treated with Z. mobilis 10 days before CLP and treatment. This was continued for 96 h after CLP induction. The animals were used for the evaluation of the survival rate. CLP model experiments were performed as previously described (Rittirsch et al., 2009). The animals were previously anesthetized intraperitoneally with a mixture of 80% ketamine and 8% xylazine. After anesthesia, a midline laparotomy was performed (exposing the cecum). This was followed by ligature and one transverse perforation (with an 18G needle) for the induction of lethal sepsis. After the surgery, the cecum of the animals was restored to its original position within the abdomen, and it was closed in two layers with 4-0 nylon sutures. Immediately after surgery, each animal received a subcutaneous injection of saline solution (1 mL). Sham-operated mice were handled in the same manner, except the cecum was not ligated or punctured. 6 h after the induction of lethal sepsis, the animals were euthanized in a CO2 chamber. The peritoneal cavity was washed with 3 mL sterile of PBS containing EDTA (3 mM). This peritoneal lavage was used to determine the total leukocytes, cytokines and quantification bacteria present. 2.4. Quantification of the total and differential leukocytes in the peritoneal cavity The peritoneal fluid was collected with a Pasteur pipette and stored in test tubes to quantify the number of total leukocytes. The counting was performed with the hematology analyzer ABX micros 60. With the aid of a cytospin SEROCITO Ò 2400, slides were made to analyze the differential leukocyte count. The slides were stained with May-Grünwald-Giemsa. The differential cell count (polymorphonuclear and mononuclear cells) was performed with an optical microscope with the aid of an immersion objective (increase of 1000 times). We counted 100 cells per slide. The results are expressed as the total number of cells (106).

2.3. Study design 2.5. Determination of cytokines in the peritoneal lavage The concentration of the bacterial suspension used in the treatments was 109 CFU/mL of Z. mobilis. This concentration was chosen in agreement with studies described in literature, which according

For the cytokine evaluation, the peritoneal lavage was centrifuged for 10 min at 2,000 rpm, and the supernatant was stored

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at 40 °C for further evaluation. The concentrations of TNF-a (a sensitivity of 8 pg/mL, and a standard curve of 8–1000 pg/mL), IL-10 (a sensitivity of 30 pg/mL, and a standard curve of 30– 4000 pg/mL), and MCP-1 (a sensitivity of 15 pg/mL, and a standard curve of 15–2000 pg/mL) were determined using ELISA according to the manufacturer’s instructions (eBioscience). The readings were taken at 450 nm. 2.6. Bacteria count in the peritoneal lavage The animals were euthanized, and after antisepsis, the peritoneal cavity was washed with sterile phosphate buffered saline (PBS). The peritoneal lavage was then diluted (1:100, 1:1000, 1:10000) in sterile PBS, and 10 uL of this solution was seeded in Petri dishes containing medium Agar Mueller Hinton (Difco Laboratories). After incubation at 32 °C for 18 h, the colonies were counted; the results were expressed as Log CFU/mL (Feng et al., 2010). 2.7. Quantification of myeloperoxidase (MPO) in lung tissue Samples of lung tissue were collected in buffer (monobasic and dibasic). Hexadecyl trimethyl ammonium bromide (HTAB) was added to the samples. The samples were then crushed with the help of a 1 polytron. The solution was adjusted to a concentration of 50 mg/mL and was centrifuged for 5 min at 6000 rpm. Then 200 lL of medium solution buffer (0.167 mg/mL of o-dianisidine 2HCl and 0.0005% H2O2) was added to each well. After 15 min of incubation at room temperature, the enzymatic reaction was interrupted with the addition of 30 lL of sodium azide (1%). The solutions were read on ELISA plates at 540 nm. Standard curves with known concentrations of myeloperoxidase (0.7–140 mU/mL) were used, allowing for the quantification of unknown values (Gomes et al., 2009).

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2.10. Statistical analysis The results were expressed as the mean ± standard deviation. Statistical analyses were performed using the Graph Pad software version 5.0 (GraphPad Software Inc, San Diego, CA, USA). We used a one-way ANOVA followed by a Bonferroni’s test, with a significance level of 0.05%. The survival of mice was evaluated using curve-Mantel Cox and log-rank tests to compare the curves. Comparison of data obtained for the enumeration of bacteria in the peritoneal cavity among groups was performed by Mann–Whitney analysis of variance. Significance was accepted at P < 0.05. 3. Results 3.1. Survival rate in lethal sepsis by CLP As shown in Fig. 1A–C, the survival of animals in the control group (C-CLP) 24 h after surgery was 12.5%. The survival decreased progressively until the end of the observation period, with 100% mortality observed. The post-treated group (T-CLP) behaved similarly to the C-CLP group, with a survival rate of 12.5% after 96 h. The early mortality was significantly delayed in pre-treated group (PT-CLP) and the prophylactic and therapeutic group (PPT-CLP). In fact, after 96 h of observation, the PT-CLP group had a survival rate of 37.5% and the PPT-CLP group had a survival rate of 50%. 3.2. Count of viable bacteria in the peritoneal lavage Previous studies show that the increase in survival was accompanied by improved control of infectious foci in animals subjected

2.8. Histopathological analyses To evaluate neutrophils migration and the extent of lung injury of mice with sepsis, animals of control group and pre-treated with Z. mobilis were sacrificed 6 h after the induction of sepsis by CLP. Fragments of the lung were removed, fixed in 10% formalin for 24 h, dehydrated in ethanol, cleared in xylene and embedded in paraffin. Fragments of 5 lm were stained with hematoxylin and eosin for histopathological analysis of the inflammatory response. 2.9. Evaluation of apoptosis of the spleen cells To evaluate the apoptosis of splenocytes in the mice submitted to lethal sepsis, spleen fragments of each group (sham, C-CLP and PT-CLP) were removed and fixed with 2.5% glutaraldehyde (Sigma) and 4% paraformaldehyde (Sigma) in 0.1 M sodium cacodylate buffer (Sigma). After fixation, the samples were washed twice in the same buffer and post-fixed in 0.1 M cacodylate buffer (pH 7.2) containing 2% osmium tetroxide (Sigma), 5 mM calcium chloride and 0.8% potassium ferrocyanide. Then the samples were dehydrated in a crescent series of acetone and included on SPIN PON-resin (Embed 812). Polymerization was performed at 60 °C for 2 days (Saraiva et al., 2006). Ultrathin sections were collected on 300mesh nickel grids, counterstained with 5% uranyl acetate and lead citrate and examined with a transmission electron microscope (Morgani FEI 268D). For each group analyzed, a minimum of 100 cells per sample were observed to evaluate cellular morphological changes. The number of apoptotic cells was expressed as a percentile.

Fig. 1. The survival rates of animals (n = 8) subjected to lethal sepsis by CLP. The data express the percentage of live animals in each group. The control group (C-CLP) (A), prophylactic and therapeutic group with Z. mobilis culture (PPT-CLP) (B) and the therapeutic group with Z. mobilis culture (T-CLP) (C) were examined. The survival rate was evaluated every 12 h for 96 h. The groups are statistically different (P 6 0.005, Mantel-Cox test).

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to sepsis by CLP. Therefore, we conducted a count of bacteria in the peritoneal lavage of the control and treated groups. The PT-CLP group showed a significant reduction in the number of bacterial CFU compared to the control group (Fig. 2). 3.3. Recruitment of neutrophils to the site of infection To investigate the underlying mechanism involved in the increase in survival of animals in the group treated prophylactically with Z. mobilis culture, we evaluated the recruitment of neutrophils to the infection site. The animals in the control group showed a marked impairment in the migration of neutrophils. The animals in the control group had four times fewer neutrophils in the peritoneal cavity compared with the group pre-treated with Z. mobilis (Fig. 3).

Fig. 4. The expression of MPO in the lung tissue of animals subjected to lethal sepsis by CLP and pre-treated with Z. mobilis culture. The expression of MPO in the lung tissue was evaluated 6 h after induction of sepsis in the animals (n = 6). The results are expressed as the mean ± SD. #P 6 0.05 when compared with the sham group. P 6 0.05 when compared with the control group (Bonferroni’s test).

3.4. Concentration of myeloperoxidase (MPO) in lung tissue Recruitment of neutrophils to the lung tissue was evaluated by MPO activity, which is an efficient marker of inflammation. As shown in Fig. 4, animals in the PT-CLP group showed a reduction in the levels of MPO compared to the control group. 3.5. Assessment of the levels of TNF-a and IL-10 after treatment with Z. mobilis It has been reported that the pro-inflammatory cytokine TNF-a, and the anti-inflammatory cytokine IL-10 have central roles in sepsis. Thus, we determined the effect of Z. mobilis on the levels of these cytokines in the peritoneal cavity. The results obtained indicate that Z. mobilis inhibited the production of TNF-a (Fig. 5A) and

Fig. 2. Evaluation of the number of bacteria in the peritoneal cavity of animals subjected to lethal sepsis by CLP. The control group (C-CLP) and the prophylactic group treated with Z. mobilis culture (PT-CLP) were examined. The bacteria present in the peritoneal cavity were evaluated 6 h after the induction of sepsis in the animals (n = 8). The results are expressed as the mean ± SD. P 6 0.05 when compared with the control group using Mann–Whitney analysis of variance.

Fig. 5. The alterations on the expression of TNF-a and IL-10 in the peritoneal fluid of animals subjected to lethal sepsis by CLP and pre-treatment with Z. mobilis culture. The control group (C-CLP) and the prophylactic group (PT-CLP) were examined. The expression of TNF-a (A) and IL-10 (B) in the peritoneal cavity was evaluated 6 h after the induction of sepsis in the animals (n = 6). The results are expressed as the mean ± SD. #P 6 0.05 when compared with the sham group.  P 6 0.05 when compared with the control group (Bonferroni’s test).

significantly increased the production of the anti-inflammatory cytokine IL-10 (Fig. 5B) compared to the control group. 3.6. Assessment of the levels of MCP-1 in peritoneal lavage The chemokine monocyte chemoattractant protein 1 (MCP-1) is a potent mediator in a wide variety of inflammatory pathologies. To assess whether Z. mobilis modulates this chemokine, concentrations of MCP-1 were assessed in the peritoneal fluid of animals subjected to CLP. However, levels of this chemokine remained similar between the control and prophylactic groups (Fig. 6). 3.7. Histopathology of the lung tissue

Fig. 3. The effect of pre-treatment with Z. mobilis culture on neutrophil migration in the peritoneal fluid of animals subjected to lethal sepsis by CLP. The control group (C-CLP) and the prophylactic group (PT-CLP) were examined. The number of neutrophils in the peritoneal cavity was evaluated 6 h after induction of sepsis in the animals (n = 5). The results are expressed as the mean ± SD. #P 6 0.05 when compared with the sham group. P 6 0.05 when compared with the control group (Bonferroni’s test).

Histological analyses showed that the control group showed alveolar thickening due to increased cellularity, cellular inflammatory cells, mild hemorrhage, congestion and emphysema (Fig. 7A– C). These results confirmed the induction of acute lung injury by this model of sepsis. Conversely, the group pre-treated with

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Fig. 6. The effect of pre-treatment with Z. mobilis culture on the expression of MCP1 in the peritoneal cavity of animals subjected to lethal sepsis by CLP. The control group (C-CLP) and the prophylactic group (PT-CLP) were examined. The expression of MCP-1 in the peritoneal cavity was evaluated 6 h after induction of sepsis in the animals (n = 6). The results are expressed as the mean ± SD. #P 6 0.05 when compared with the sham group (Bonferroni’s test).

Z. mobilis showed up better aspect of the morphology of the lung parenchyma when compared to the control group (Fig. 7D–F). 3.8. Ultrastructural analyses of splenocytes The ultrastructural analyses of the splenocytes of the sham group showed a typical morphology pattern (a rounded nucleus with prominent heterochromatin, rough endoplasmic reticulum mitochondria, and glycogen granules) (Fig. 8E and F). Apoptosis of the immune cells plays a central role in the pathophysiology of sepsis. Prevention of apoptosis in one cell type can confer benefits to the neighboring cells (Chang et al., 2007). The control group showed that 83% of the splenocytes had morphological changes (condensed chromatin and apoptotic bodies) (Fig. 8A and B). However, animals submitted to prophylactic treatment with Z. mobilis presented several well-preserved cells. Only 24% of the cells presented morphological characteristic of apoptosis, similar to that observed in the sham group, which presented with only 19% apoptotic splenocytes (Fig. 8C and D). 4. Discussion The results of the present study show that pretreatment with Z. mobilis can decrease the mortality rate, attenuate lung injury

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resulting from CLP, up-regulate the levels of the anti-inflammatory cytokine IL-10 and enhance the ability to control infection by reducing the number of bacteria. To further explore the mechanisms underlying Z. mobilis-mediated protection against polymicrobial sepsis, we investigated the effects of Z. mobilis on the bacterial burden. Our results demonstrate that there was better control of the infection, as shown by a pronounced decrease in CFUs at the infection site. These data agree with the data presented by other authors (Benjamim et al., 2002; Crosara-Alberto et al., 2002). In the CLP model, the bacteria spread from infection sites by entering the bloodstream. The bacteria are rapidly trapped in many organs such as the liver, lung, kidney, and spleen. These bacteria are bound to the surface of specific target cells and macrophages in the target organ (Gregory et al., 1992). The organs in mice are impaired after lethal sepsis induced by CLP. This impairment is associated with an ineffective bacterial clearance, leading to bacterial dissemination and high mortality rates (Moreno et al., 2006). In our study, administration of a Z. mobilis culture led to a marked increase in survival, which was at least partially associated with an increased bacterial clearance. The probiotic properties of Z. mobilis CP4 (UFPEDA-202) was studied in a wistar rat fed the 109 colony forming units (CFU)/ mL, and were observed that no bacteria were found in blood, liver and spleen of animals, indicating no bacterial translocation (Azerêdo et al., 2010). Therefore, we can infer that the increased survival of animals with sepsis observed in our study may be related to the direct antagonism exerted by Z. mobilis against pathogenic bacteria well as protection of the intestinal mucosa, reducing the spread of pathogenic bacteria. However, the mode of action of probiotics is complex and not yet fully elucidated. Many mechanisms have been reported to explain probiotic actions such as antagonism against intestinal pathogens, enhancement of mucosal barrier activity, or modulation of host’s immune functions as recently reviewed (Gareau et al., 2010). Sepsis is characterized by an initial net hyperinflammatory response, followed by a period of immunosuppression that has been termed ‘‘immunoparalysis’’. This immunosuppression in sepsis is characterized by numerous defects in both the innate and adaptive immune system. This is also associated with failure of polymorphonuclear leukocyte migration (Monneret 2005; Murphy et al., 2004; Remick 2007). It is known that local migration of neutrophils

Fig. 7. Histopathological analyses of the lungs of animals subjected to sepsis by CLP and pre-treated with Z. mobilis culture. A–C: Animals with sepsis by CLP (control group), there is intense interstitial reaction, with marked inflammation, reduction in alveolar spaces and thickening of the septum, and vascular congestion (A and B) and C, can observe the presence of neutrophils, eosinophils and mononuclear cells (monocytes). D–F: Animals pretreated with Z. mobilis, shows up better aspect of the morphology of the lung parenchyma when compared to the control group. HE: A and D (100), B and E (400) and C and F (1000 with immersion oil).

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Fig. 8. The apoptosis of splenocytes in animals subjected to sepsis by CLP and pre-treatment with Z. mobilis. Animals in the control group showed a condensation of nuclear chromatin and apoptotic bodies (A and B). The animals that received prophylactic treatment with Z. mobilis (C and D) as well the sham group (E and F) presented with preserved splenocytes. A, C and E were viewed under 3500 magnification. B, D and F were viewed under 7500 magnification.

to the injury site is very important to repair damaged tissue and to protect against invading microorganisms (Ronco et al., 2011). Our findings reveal a major failure of neutrophil migration in the control group. This is in contrast to the animals in the treated group (treated prophylactically with Z. mobilis), where there was an increase in cell migration, improving the dysfunction that occurs in sepsis. Studies confirm that the impairment of neutrophil migration toward an infectious focus in animals subjected to severe sepsis is associated with a large deficit in the control of infection. This event leads to greater systemic dissemination of bacteria, as well as a high mortality rate (Benjamim et al., 2000; Maciel et al., 2008; Torres-Dueñas et al., 2007). Acute lung injury is a complication of sepsis and is associated with high morbidity and mortality in patients with sepsis (Chopra et al., 2009). Approximately 20% of patients exhibiting severe sepsis develop lung dysfunction (Lucas 2007). In the polymicrobial sepsis model, a slight lung injury (characterized by the coagulation cascade) occurs at 24 h post-cecal ligation and puncture (CLP). This injury is associated with capillary congestion and perivascular cuffing, along with increased lung myeloperoxidase activity, an

index of neutrophil sequestration (Mercer-Jones et al., 1997). The maximum activity of MPO in the lungs is found 6 h after induction of sepsis by CLP. The accumulation of activated PMNs in the lung tissue during severe lung inflammation suggests that these cells play a significant role in the development of acute lung injury (Speyer et al., 2004). In this study, we observed a reduction in MPO levels in the lung tissue of animals treated with Z. mobilis, confirming the histopathological findings. He et al. (2009) studied the action of the chemokine fractalkine. The study concluded that injection of this chemokine in animals before induction of sepsis by CLP reduced lung injury. This reduction in lung injury resulted from a decrease in the concentration of MPO compared to the control group. In another study, the use of methylene chloride in the treatment of sepsis induced by CLP reduced the MPO levels in lung tissue, improved the rate of survival, increased the levels of IL-10 and reduced the levels of TNF-a (Pang et al., 2010). Our findings regarding Z. mobilis treatment agree with these studies. We show a reduction in the concentration of MPO in the lung tissue, which was confirmed by the results of the histopathological evaluation. While the animals in the control group exhibited histopathological

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features consistent with an acute lung injury, animals that received pre-treatment with Z. mobilis showed improvement in this regard. The rapid innate immune response generates high levels of inflammatory cytokines and constitutes the first line of self-defense against bacterial infections. However, uncontrolled production of cytokines is a major cause of septic death (Inoue et al., 2010). To understand the mechanisms involved in protection from CLP lethality by Z. mobilis, we assessed the inflammatory cytokine production in the CLP-treated and control groups. In our study, there was marked increase in the concentration of IL-10 and a decrease in the concentration of TNF-a. However, the levels of MCP were not altered. TNF-a is one of the mediators responsible for organ dysfunction and increased mortality during severe sepsis (Gaur and Aggarwal, 2003; Gordon et al., 2004). Studies show that the increase in the survival rate is accompanied by decreased levels of TNF-a (Alves-Filho et al., 2010; Ma et al., 2006; Tavares-Murta et al., 1998). Excessive production of pro-inflammatory cytokines and suppression of anti-inflammatory cytokines can induce a decrease in the number of neutrophils recruited to the lesion site (Gordon et al., 2004). Controversy exists concerning the role of interleukin 10 (IL-10) in sepsis. When IL-10 is used in models of endotoxemia, it appears to be protective (via its anti-inflammatory effects). However, in models of polymicrobial sepsis, it seems to be deleterious (Song et al., 1999). IL-10 is a cytokine responsible for wide immunoregulatory ability, acting both on the innate immune system and the adaptive immune system (Lindsay and Hodgson 2001). This immunomodulatory activity is based on its ability to inhibit the synthesis of chemokines responsible for recruitment of cells. IL-10 can also induce a systemic inflammatory syndrome, which occurs in sepsis (Moore et al., 2001). Cao et al. (2010) attributed the protective role of ulinastatin (in a murine model of sepsis induced by CLP) to the increase in the intestinal levels of IL-10 and IL-8 in the study animals. In our study, the marked increase in the concentration of IL-10 could modulate the concentrations of TNFa, thus increasing the survival of animals. Accumulating evidence suggests a pivotal role for apoptosis in sepsis-induced immunosuppression (Ayala et al., 2008; Lang and Matute-Bello, 2009; Ward 2008). Studies have shown that the numbers of peripheral and splenic lymphocytes are reduced during sepsis in both humans and animals (Hotchkiss et al., 2001). Apoptosis is known to be responsible for decreased lymphocyte numbers, and the extent of lymphocyte apoptosis correlates with the severity of sepsis (Wesche et al., 2005). During sepsis, deregulation of the lymphocytes leads to apoptosis in the thymus, spleen and GALT. These events can lead to immune suppression, leaving the patient vulnerable and resulting in multiple organ failure (Hotchkiss et al., 2001). Apoptotic cells may induce an anergy that harms the host, as shown by Hotchkiss et al. (2003). This study found that adoptive transfer of apoptotic splenocytes in septic mice substantially increased mortality. Our findings show that animals in the group treated prophylactically with Z. mobilis showed a marked reduction in the rate of apoptotic splenocytes when compared to the control group. This suggests that Z. mobilis can exert immunomodulatory actions. Further studies by our group are underway, which are aimed at better understanding the underlying mechanism(s) of Z. mobilis. To our knowledge, this is the first report detailing the effects of Z. mobilis culture in sepsis. Taken together, our results show that Z. mobilis can have immunomodulating actions through the regulation of cytokines and improvement in neutrophil migration, substantially reducing acute lung injury and decreasing the apoptosis of lymphocytes present in the spleen. Furthermore, there is evidence that Z. mobilis presented antagonism against intestinal pathogens, and enhancement of mucosal barrier activity. Therefore, this non-pathogenic bacterium is a promising candidate to treat or prevent infectious diseases.

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