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Long-term effects of neonatal malnutrition on microbicide response, production of cytokines, and survival of macrophages infected by Staphylococcus aureus sensitive/resistant to methicillin

Efeitos tardios da desnutrição neonatal na resposta microbicida, produção de citocinas e viabilidade de macrófagos na infecção por Staphylococcus aureus sensível/resistente a meticilina

Abstracts

OBJECTIVE:

To assess microbicide function and macrophage viability after in vitro cellular infection by methicillin-sensitive/resistant Staphylococcus aureus in nourished rats and rats subjected to neonatal malnutrition.

METHODS:

Male Wistar rats (n=40) were divided in two groups: Nourished (rats suckled by dams consuming a 17% casein diet) and Malnourished (rats suckled by dams consuming an 8% casein diet). Macrophages were recovered after tracheotomy, by bronchoalveolar lavage. After mononuclear cell isolation, four systems were established: negative control composed exclusively of phagocytes; positive control composed of macrophages plus lipopolysaccharide; and two testing systems, macrophages plus methicillin-sensitive Staphylococcus aureus and macrophages plus methicillin-resistant Staphylococcus aureus. The plates were incubated in a humid atmosphere at 37 degrees Celsius containing 5% CO2 for 24 hours. After this period tests the microbicidal response, cytokine production, and cell viability were analyzed. The statistical analysis consisted of analysis of variance (p<0.05).

RESULTS:

Malnutrition reduced weight gain, rate of phagocytosis, production of superoxide anion and nitric oxide, and macrophage viability. Production of nitrite and interleukin 18, and viability of macrophages infected with methicillin-resistant Staphylococcus aureus were lower.

CONCLUSION:

The neonatal malnutrition model compromised phagocyte function and reduced microbicidal response and cell viability. Interaction between malnutrition and the methicillin-resistant strain decreased the production of inflammatory mediators by effector cells of the immune response, which may compromise the immune system's defense ability.

Macrophages; Malnutrition; Methicillin; Staphylococcus


OBJETIVO:

Avaliar a função microbicida e a viabilidade de macrófagos, após infecção celular in vitro, com Staphylococcus aureus sensível/resistente a meticilina, em ratos nutridos ou submetidos a desnutrição neonatal.

MÉTODOS:

Ratos machos Wistar (n=40) foram divididos em dois grupos distintos: Nutrido (ratos amamentados por mães submetidas a dieta com 17% de caseína) e Desnutrido (ratos amamentados por mães submetidas a dieta com 8% de caseína). Os macrófagos foram recuperados após procedimento cirúrgico de traqueostomia, através da coleta do lavado broncoalveolar. Após o isolamento dos mononucleares, foram estabelecidos quatro sistemas: controle negativo, composto apenas pelos fagócitos; controle positivo, macrófagos mais lipopolissacarídeo; e dois sistemas teste, macrófagos mais Staphylococcus aureus sensível e resistente a meticilina. As placas foram incubadas por 24 horas, à temperatura de 37ºC, com atmosfera úmida e 5% de dióxido de carbono. Transcorrido esse período, foram realizados ensaios para análise da resposta microbicida, produção de citocinas e viabilidade celular. Na análise estatística, utilizou-se analysis of variance, admitindo-se p<0,05.

RESULTADOS:

A desnutrição acarretou redução do crescimento ponderal dos animais, da taxa de fagocitose, da produção de óxido nítrico, do ânion superóxido e da viabilidade de macrófagos. Houve menor produção de nitrito, de interleucina 18 e da viabilidade dos macrófagos infectados com Staphylococcus aureus meticilina-resistente.

CONCLUSÃO:

O modelo de desnutrição neonatal adotado comprometeu a função dos fagócitos, com redução da resposta microbicida e da viabilidade celular. A interação de desnutrição com cepa resistente induziu baixa produção de mediadores inflamatórios por células efetoras da resposta imunológica, o que poderá resultar em compro-metimento da defesa.

Macrófagos; Desnutrição; Meticilina; Staphylococcus


INTRODUCTION

Environmental insults during vulnerable periods of an organism's development can permanently affect the structure and function of organs and tissues. This vulnerability is associated with the intense differentiation and maturation that organ systems undergo during these periods11. Pereira KNF, Vitoriano ILS, Melo MPP, Aragão RS, Toscano AE, Silva HJ, De Castro RM. Effects of malnutrition and/or neonatal inhibition of serotonin reuptake in neuromuscular development of the gastrointestinal tract: Review of literature. Neurobiology. 2009; 72(2):215-21.. Studies have shown that neonatal malnutrition can compromise the formation of homeostatic systems, such as the nervous, endocrine, and immune systems22. Chandra RK. Nutrition and the immune system from birth to old age. Eur J Clin Nutr. 2002; 56(3):73-6. doi: 10.1038/sj.ejcn.1601492
https://doi.org/10.1038/sj.ejcn.1601492...
, 33. Melo JF, Costa TC, Lima TDC, Chaves MEC, Vayssade LM, Nagel MD, et al. Long-term effects of a neonatal low-protein diet in rats on the number of macrophages in culture and the expression/production of fusion proteins. Eur J Nutr. 2013; 52(5):1475-82. doi: 10.1007/s00394-012-0453-y
https://doi.org/10.1007/s00394-012-0453-...
.

According to Chandra22. Chandra RK. Nutrition and the immune system from birth to old age. Eur J Clin Nutr. 2002; 56(3):73-6. doi: 10.1038/sj.ejcn.1601492
https://doi.org/10.1038/sj.ejcn.1601492...
, neonatal malnutrition affects the macrophages' functional mechanism, causing enduring changes in the adult organism, even long after nutritional recovery. Malnourished individuals may have deficient phagocytic microbicidal function, such as low production of proinflammatory cytokines, free radicals (nitric oxide and superoxide anion), and macrophage viability, making the body more vulnerable to infections44. Melo JF, Macedo EMC, Silva RPP, Viana MT, Silva WTF, Castro CMMB. Efeito da desnutrição neonatal sobre o recrutamento celular e a atividade oxidante--antioxidante de macrófagos em ratos adultos endotoxêmicos. Rev Nutr. 2008; 21(6):683-94. doi: 10.1590/S1415-52732008000600007
https://doi.org/10.1590/S1415-5273200800...
, 55. Costa TB, Morais NG, Almeida TM, Severo MS, Castro CMMB. Early malnutrition and production of IFN-?, IL-12 and IL-10 by macrophages/lymphocytes: In vitro study of cell infection by methicillin-sensitive and methicillin-resistant Staphylococcus aureus. Rev Nutr. 2012; 25(5):607-19. doi: 10.1590/S1415-52732012000500006
https://doi.org/10.1590/S1415-5273201200...
.

Pathogenic invasion may deregulate the microbicidal responsiveness of immune66. Tegnér J, Nilsson R, Bajic VB, Björkegren J, Ravasi T. Systems biology of innate immunity. Cell Immunol. 2007; 244(2):105-9. doi: 10.1016/j.cellimm.2007.0 1.010
https://doi.org/10.1016/j.cellimm.2007.0...
cells. This mechanism is triggered by various microorganisms to facilitate their growth and increase their survival time in the host77. Kubica M, Guzik K, Koziel J, Zarebski M, Richter W, Gajkowska B, et al. Potential new pathway for Staphylococcus aureus dissemination: The silent survival of S. aureus Phagocytosed by human monocyte-derived macrophages. PLOS ONE. 2008; 3(1):1409-35. doi: 10.1371/journal.pone.0001409
https://doi.org/10.1371/journal.pone.000...
. In order to establish an infection, Staphylococcus aureus has developed several mechanisms designed to withstand the immune response.

Since the 1960's, infection by Methicillin-Resistant Staphylococcus Aureus (MRSA) has been considered a public health problem worldwide, mainly because it is more deadly than Methicillin-Sensitive Staphylococcus Aureus (MSSA)88. Beam JW, Buckley B. Community-acquired methicillin-resistant Staphylococcus aureus: Prevalence and risk factors. J Athl. 2006; 41(3):337-40.. MRSA strains seem to have different virulence mechanisms, more intense than MSSA strains99. Mandell GL. Uptake, transport, delivery and intracellular activity of antimicrobial agents. Pharmacother. 2005; 25(12):130S-3S.. Numerous clinical studies on morbidity and mortality rates have indicated that MRSA is more virulent than MSSA. However, laboratory studies that assessed the presence and magnitude of pathogenic mechanisms and virulence factors in MSSA and MRSA strains obtained conflicting results55. Costa TB, Morais NG, Almeida TM, Severo MS, Castro CMMB. Early malnutrition and production of IFN-?, IL-12 and IL-10 by macrophages/lymphocytes: In vitro study of cell infection by methicillin-sensitive and methicillin-resistant Staphylococcus aureus. Rev Nutr. 2012; 25(5):607-19. doi: 10.1590/S1415-52732012000500006
https://doi.org/10.1590/S1415-5273201200...
, 1010. Morais NG, Costa TB, Almeida TM, Severo MS, Castro CMMB. Parâmetros imunológicos de macrófagos frente à infecção por Staphylococcus aureus meticilina sensível/resistente. J Bras Patol Med Lab. 2013; 49(2):84-90. doi: 10.1590/S1676-24 442013000200002
https://doi.org/10.1590/S1676-2444201300...
.

Despite the various studies using malnutrition models, there is still a lot to be researched in terms of neonatal malnutrition and its late effects on the immune response. Also, there are hardly any studies evaluating the interaction between infection and malnutrition. The present study aimed to evaluate the impact of neonatal malnutrition on microbicidal function, cytokine production, and viability of alveolar macrophages infected in vitro by methicillin-sensitive and MRSA. In this context animal experiments may help to clarify morphological changes in early life stages, their intimate relation with microbicidal response, and infectious disease emergence and evolution.

METHODS

Animals and diet

Forty male Wistar rats (90-120 days) from the Universidade Federal do Pernambuco (UFPE) Department of Nutrition animal facility were used for the experiments. The experiments were conducted ethically as recommended by the Colégio Brasileiro de Experimentação Animal (COBEA, Brazilian College of Animal Experimentation) and the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of the Center for Biological Sciences, UFPE under Protocol nº 23076.026684/2009-38. The animals were kept under controlled temperature (22°C, Standard Deviation-(SD)=1°C) in 12:12 hours light-dark cycles and had free access to water and chow.

One day after birth, litter size was standardized to six male pups per mother. On the same day, their first day of life, the litters were divided into two groups: a Nourished (N) group consisting of pups nursed by dams consuming a 17% casein diet (n=20); and a Malnourished (M) group consisting of pups nursed by dams consuming an 8% casein diet (n=20) for the first 21 days after birth. The 8% casein diet is widely used as an experimental model for the study of malnutrition because it induces protein malnutrition1111. Passos MCF, Ramos CF, Moura EG. Short and long term effects of malnutrition in rats during lactation on the body weight of offspring. Nutr Res. 2000, 20(1):1603-12. doi: 10.1016/S0271-5317(00)00 246-3
https://doi.org/10.1016/S0271-5317(00)00...
in animals.

During the neonatal period the animals were weighed daily by a digital electronic scale (Marte, model S-4000, with an accuracy of 0.1 g) to monitor their body weight during nutritional manipulation. From the 22nd day of life until the end of the experiment, the animals were weighed three times a week to monitor their nutritional recovery. During this period the animals were separated from the dams, kept in cages in groups of three, and fed a standard chow (Vivarium, Labina Purina-Brazil) until adulthood (approximately 90 days).

Strains of Staphylococcus aureus

Strains of Methicillin-Sensitive Staphylococcus Aureus (The American Type Culture Collection - ATCC 33591) and methicillin-sensitive Staphylococcus aureus were used due to their distinctive resistance and importance in terms of public health. The bacteria were maintained in Tryptic Soy Broth (TSB) supplemented with 20% glycerol, at -20°C, until use. Twenty-four hours before each experiment, the strains were plated on blood agar (agar supplemented with 5% sheep blood) and incubated at 37°C. At the beginning of the test, some colonies were transferred to tubes containing Phosphate-Buffered Saline (PBS) to provide a turbidity of approximately 0.15 nm at a wavelength of 570 nm. This absorbance, according to Lu & McEwan1212. Lu YF, McEwan NA. Staphylococcal and micrococcal adherence to canine and feline corneocytes: Quantification using a simple adhesion assay. Veterinary Dermatol. 2007; 18(1):29-35. doi: 10.1111/j.1365-3164.2007.00567
https://doi.org/10.1111/j.1365-3164.2007...
, corresponds to a concentration of approximately 106 6. Tegnér J, Nilsson R, Bajic VB, Björkegren J, Ravasi T. Systems biology of innate immunity. Cell Immunol. 2007; 244(2):105-9. doi: 10.1016/j.cellimm.2007.0 1.010
https://doi.org/10.1016/j.cellimm.2007.0...
bacteria/mL of PBS.

Bronchoalveolar lavage

Bronchoalveolar Lavage (BAL) was done as recommended by Castro et al. 1313. Castro CMMB, Manhães-de-Castro R, Medeiros AF, Santos AQ, Silva WTF, Lima Filho JLL. Effect of stress on the production of O2(- ) in alveolar macrophages. J Neuroimmunol. 2000; 108(1-2):68-72.. The animals were anaesthetized with chloralose-urethane (0.5 and 12.5%, respectively) at 8 mL/kg ip. BAL was collected by injecting 0.9% saline through a plastic cannula into the trachea. Several aliquots of 3 mL were then injected and collected in 50 mL conical polypropylene tubes (Falcon, Sigma).

Culture of alveolar macrophages

Bronchoalveolar Lavage samples were centrifuged at 1500 rpm for 15 minutes. The precipitate that corresponds to the cells was resuspended in RPMI 1640 (Roswell Park Memorial Institute medium - Gibco, Invitrogen Corporation) containing 3% fetal bovine serum (Gibco-Invitrogen Corporation) and antibiotics (100 U penicillin/mL and 100 μg streptomycin/mL).

The cells were transferred to 35 mm diameter (6-well Falcon) cell culture dishes, in which a 2 mL suspension was dispensed in a proportion of 1066. Tegnér J, Nilsson R, Bajic VB, Björkegren J, Ravasi T. Systems biology of innate immunity. Cell Immunol. 2007; 244(2):105-9. doi: 10.1016/j.cellimm.2007.0 1.010
https://doi.org/10.1016/j.cellimm.2007.0...
cells/mL in RPMI 1640. After 1 hour in an incubator at 37°C and 5% CO2, the supernatant was discarded with non-adherent cells and 2 mL of half RPMI were added, leaving the plates for another 1 hour in the incubator in order to stabilize the cells.

Systems

Three systems were established in order to evaluate the rate of adherence, phagocytosis, and superoxide production: Control (C), with only alveolar macrophages; MSSA, Alveolar Macrophages (AM) plus 100 µL of bacterial inoculum on the methicillin-sensitive strain (ATCC 29213); and MRSA, AM plus 100 µL of bacterial inoculum of methicillin-resistant strain (ATCC 33591). To evaluate the kinetics of nitric oxide and alveolar macrophage viability, a positive control was added - PC containing AM plus 10 μL of Lipopolysaccharide (LPS) (Escherichia coli serotype; 055: B5, Sigma). Subsequently, the plates were incubated at 37°C in a wet atmosphere containing 5% CO2.

Assessment of the adhesion rate

After incubation of cell cultures for 1 hour, aliquots were collected from the supernatant containing non-adherent cells and wells of the filtration plate were resuspended with RPMI. These aliquots containing non-adherent cells were added to trypan blue stain (1:10 dilution) and cells were counted using a hemocytometer. The Adhesion Rate (AR) was calculated using the formula described by De la Fuente et al. 1414. De la Fuente M, Del Rio M, Ferrandez MD, Hernanz A. Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C. Immunology. 1991; 73(2):205-11.: AR=100 - non adhered cells/mL/the initial number of cells/mL x 100.

Determination of the rate of phagocytosis

For this assay the bacterial inoculum was added to a suspension of 1066. Tegnér J, Nilsson R, Bajic VB, Björkegren J, Ravasi T. Systems biology of innate immunity. Cell Immunol. 2007; 244(2):105-9. doi: 10.1016/j.cellimm.2007.0 1.010
https://doi.org/10.1016/j.cellimm.2007.0...
cells/mL in RPMI 1640 for both strains to a concentration of 1066. Tegnér J, Nilsson R, Bajic VB, Björkegren J, Ravasi T. Systems biology of innate immunity. Cell Immunol. 2007; 244(2):105-9. doi: 10.1016/j.cellimm.2007.0 1.010
https://doi.org/10.1016/j.cellimm.2007.0...
CFU/mL in PBS, with a remainder volume of 1.5 mL in each tube. The contents of the tubes were homogenized and gently/evenly distributed on slides for optical microscopy. Slides were placed in an oven for 1 hour. After this period, they were washed to remove non-adherent cells and then stained and viewed by a trained, "blind" observer (for the system under analysis) using a light microscope. The result was expressed in percentage of phagocytic cells in a total count of 100 cells1515. Malagueno E, Albuquerque C, Castro CMMB, Gadelha M, Inácio-Irmão J, Santana JV. Effect of biomphalaria straminea plasma of biomphalaria glabrata hemolymph cells. Mem Inst Oswaldo Cruz. 1998; 93(1):301-2. doi: 10.1590/S0074-027619 98000700059
https://doi.org/10.1590/S0074-0276199800...
.

Analysis of low superoxide anion (O2-) release

O2 - was induced by adding phorbol myristate acetate/PMA (Sigma) to Hank's solution (HBSS, Gibco-Invitrogen Corporation(r)) at a concentration of 2 μg/mL. Analysis of discontinuous systems was prepared with hourly assessments for 2 hours. Assay specificity was confirmed by the addition of Superoxide Dismutase (SOD) from bovine erythrocytes, containing 3000 U/mg protein in the final solution of 3 mg/mL in distilled water (Sigma)1313. Castro CMMB, Manhães-de-Castro R, Medeiros AF, Santos AQ, Silva WTF, Lima Filho JLL. Effect of stress on the production of O2(- ) in alveolar macrophages. J Neuroimmunol. 2000; 108(1-2):68-72..

Kinetics of nitric oxide production by alveolar macrophages

The production of NO was given by the concentration of nitrite in the culture supernatant1010. Morais NG, Costa TB, Almeida TM, Severo MS, Castro CMMB. Parâmetros imunológicos de macrófagos frente à infecção por Staphylococcus aureus meticilina sensível/resistente. J Bras Patol Med Lab. 2013; 49(2):84-90. doi: 10.1590/S1676-24 442013000200002
https://doi.org/10.1590/S1676-2444201300...
. Every two hours, 100 µL of the supernatant were taken from the cultures, in a total incubation period of 24 hours. To quantify nitrates and nitrites, 50 mL of Griess reagent (1.5% sulfanilamide in 5% H3PO4, 0.1% in N-(1-naphthyl)ethylenediamine H20) were added to supernatants. After standing 15 minutes at room temperature, an reader Enzyme Linked Assay (ELISA, ImmunonoSorbent - BIO-RAD, Model 680), with 550 nm filter, was used for the procedure. The nitrite concentration was calculated using average values of a NaNO2 standard curve, data expressed in µM.

Quantification of cytokines IL-1β

and IL-18 (interleukin-1β and

interleukin-18)

After 24 hours of cell culture incubation, 100 µL of supernatant were collected. From this, IL-1β and IL-18 cytokines were counted by ELISA immunoenzyme test, using the Quantikineâ m (R&D Systems) kit.

Viability of alveolar macrophages

Cell viability was assessed by mitochondrial reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) on formazan1616. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immun Meth. 1983; 65(1/2):55-63.. After 24 hours of incubation, cell cultures were washed with PBS (1X) at room temperature They were incubated with 550 µL of PBS and 55 µL of MTT solution for two hours protected from light. After this period, 200 µL of PBS and 200 µL of DMSO were added and the cell monolayer was scraped. Quantification of solubilized formazam was performed in an ELISA reader (Bio-Rad, model 680) with 570 nm filter. Results were expressed in absorbance of formazan (1x1066. Tegnér J, Nilsson R, Bajic VB, Björkegren J, Ravasi T. Systems biology of innate immunity. Cell Immunol. 2007; 244(2):105-9. doi: 10.1016/j.cellimm.2007.0 1.010
https://doi.org/10.1016/j.cellimm.2007.0...
cells).

Statistical analysis

The Student's t-test was used for the body weight and the rate of phagocytosis analysis. The other parametric data were analyzed by Analysis of Variance (Anova). When Anova revealed a significant difference, the Tukey test was used for identifying which groups differed from each other. The significance level was set at p<0.05. The statistical program used for the analyses was Sigma Stat 3.5 version.

RESULTS

Body weight on malnutrition

and nutritional recovery

The body weights (g) of the nourished and malnourished groups were similar until the 3rd day of life. From the 4th to the 21st postnatal day, the malnourished animals were lighter than the nourished animals (p<0.001). Between the 22nd and 90th days of life the two groups were fed the same chow, but the malnourished group remained lighter than the nourished group (p<0.001) (Figure 1).

Figure 1.
Weight curve during the neonatal malnutrition period (21 days) and nutritional supplementation period (23-90 days) of the groups (N: Nourished- and M: Malnourished). Recife (PE), 2009. Note: (*)p<0.05 in comparison between Nourished and Malnourished groups. Student's t test. Values are expressed as Mean ± Standard Deviation (n=40).

Grip index

There were no differences between the nourished and malnourished groups (C-N=87.5±3,0; C-M=86±2,0; MSSA-N=90.3±2.6; MSSA-M=91±2.0; MRSA-N=93.3±3.1; MRSA=93±3.0), p>0.05. Also, no differences were observed between the systems under analysis, p>0.05.

Rate of phagocytosis

The rate of phagocytosis was lower in the malnourished group (MSSA-N=12.1±2.0; MSSA-M=4.1±3.2; MRSA-N=10.4±3.1; MRSA-M=4.3±3.0), p<0.001. However, when analyzing the MSSA and MRSA systems, there was no difference in the rates of phagocytosis of alveolar macrophages (p>0.05).

Production of superoxide

The malnourished group produced less superoxide than the nourished group for all systems (p<0.001) for both incubation times (p<0.05). However, the MSSA and MRSA systems did not differ (p>0.05) (Table 1).

Table 1.
Absolute figures for superoxide production in the groups (N:Nourished; M:Malnourished), in the systems (C: Negative Control; MSSA: Methicillin-Sensitive Staphylococcus Aureus; MRSA: Methicillin-Resistant Staphylococcus Aureus) and incubation periods (1 and 2 hours). Recife (PE), 2009.

Kinetic analysis of the nitric oxide production by alveolar macrophages

The quantification of NO was expressed in µM nitrite. The production of NO by macrophages was lower in malnourished animals in all systems (p<0.05). Differences were found between NC and PC systems in both groups after 8 hours of incubation, with high production of nitric oxide in the PC (p<0.001). The peak NO production for the PC system occurred after 22 hours, both for the nourished and the malnourished group (p<0.001). From 4 to 10 hours, the average NO production was lower than in the nourished group in the malnourished group in the MSSA and MRSA systems (p<0.05). The peak NO production occurred after a 4h incubation period in MSSA system, group N, after 6h for group D, and after 8h for MRSA in both groups. Up to 12 hours of incubation, both for the nourished and malnourished groups, there was a reduction of NO production for MSSA and MRSA systems (p<0.001), similar to those of CN (Figure 2).

Figure 2.
Nitric oxide production in the supernatant of alveolar macrophage cultures in groups (N: Nourished; M: Malnourished) and systems (PC: Positive Control, MSSA: Methicillin Sensible Staphylococcus Aureus; MRSA: Methicillin Resistant Staphylococcus Aureus). Recife (PE), 2009. Note: (*)p<0.05 on the comparison of the Nourished and Malnourished groups. Analysis of Variance and Tukey test. Values are expressed as Mean ± Standard Deviation (n=40).

IL-1β Levels

The levels of IL-1β of nourished versus malnourished controls did not differ (p>0.05). However, when analyzing the testing systems, there was a lower concentration of IL-1β in the supernatant of the MRSA testing systems (MSSA-N=15.94±0.53 pg/mL; MSSA-M=11.81±3.01 pg/mL; MRSA-N=4.24±0.26 pg/mL; MRSA-M=6.41±0.3 pg/mL) p<0.05 (Figure 3).

Figure 3.
Levels of IL-1â in the supernatant of alveolar macrophage cultures in groups (N: Nourished; M: Malnourished) and systems (C: Negative Control, MSSA: Methicillin Sensible Staphylococcus Aureus; MRSA: Methicillin Resistant Staphylococcus Aureus). Recife (PE), 2011. Note: *p<0.05 in comparison of Nourished and Malnourished groups. Analysis of Variance and Tukey test. Values are expressed as Mean ± Standard Deviation (n=40).

IL-1β Levels

The levels of IL-18 of nourished versus malnourished controls did not differ (p>0.05). The production of IL-18 was higher in MSSA (MSSA-N=5.87±0.59 pg/mL; MRSA-N=3.11±0.23 pg/mL) p<0.05. However, when analyzing the testing systems, there was a lower concentration of IL-18 in MRSA testing systems of the malnourished group (MRSA-N=3.11±0.23 pg/mL; MRSA-M=0.27±0.01 pg/mL) p<0.05 (Figure 4).

Figure 4.
IL-18 levels in the supernatant of alveolar macrophage cultures in groups (N: Nourished; M: Malnourished) and systems (C: Negative Control, MSSA: Methicillin Sensible Staphylococcus Aureus; MRSA: Methicillin Resistant Staphylococcus Aureus). Recife (PE), 2011. Note: (*)p<0.05 in comparison of Nourished and Malnourished groups. Analysis of Variance and Tukey test. Values are expressed as Mean ± Standard Error Deviation (n=40).

Viability of alveolar macrophages

The malnourished group had lower macrophage viability than the nourished group in all systems under analysis (PC-N=69.2±0.8; PC-M=51.3±0.51; MSSA-N=18.5±0.22; MSSA-M=12.2±0.11; MRSA-N=20.7±0.2; MRSA-M=6.7±0.03), p<0.05. The number of macrophages in the MRSA system of the malnourished group was significantly smaller (p<0.001).

DISCUSSION

Studies show that neonatal malnutrition models correlate with the deficiency of certain nutrients and gene expression, changing the genotype and phenotype1717. Rodriguez L, Gonzalez C, Flores L, Jiménez-Zamudio L, Graniel J, Ortiz R. Assessment by flow cytometry of cytokine production in malnourished children. Clin Diagn Lab Immunol. 2005; 12(4):502-7. doi: 10.1111/j.1365-2249.2007.03361
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, 1818. Waterland RA, Jirtle RL. Early nutrition, epigenetic changes at transposons and imprinted genes, and enhanced susceptibility to adult chronic diseases. Nutrition. 2004; 20(1):63-8. doi: 10.1016/j.nut.20 03.09.011
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. The intensity and duration of malnutrition will determine the extent of systemic consequences11. Pereira KNF, Vitoriano ILS, Melo MPP, Aragão RS, Toscano AE, Silva HJ, De Castro RM. Effects of malnutrition and/or neonatal inhibition of serotonin reuptake in neuromuscular development of the gastrointestinal tract: Review of literature. Neurobiology. 2009; 72(2):215-21..

In this study the experimental model of malnutrition consisted of an 8% casein diet, which is considered low protein. The low protein level on the diet offered to dams is characterized by the restricted amount of nutrients available to puppies. Thus, infants develop protein malnutrition, while puppies develop protein-calorie malnutrition. This fact is crucial for the genesis of the deleterious effects observed in the offspring1919. Araujo FRG, De Castro CMMB, Rocha JA, Sampaio B, Diniz MFA, Evêncio LB, et al. Perialveolar bacterial microbiota and bacteraemia after dental alveolitis in adult rats that had been subjected to neonatal malnutrition. Br J Nutr. 2012; 107(1):996-1005. doi: 10.1017/S00 0711451100393X
https://doi.org/10.1017/S000711451100393...
.

The animals nursed by these dams were stunted, evidenced by low weight at weaning that persisted to 90 days of age. From the fourth postnatal day, malnourished animals gained less weight than the nourished samples. This result is similar to that found by Melo et al. 44. Melo JF, Macedo EMC, Silva RPP, Viana MT, Silva WTF, Castro CMMB. Efeito da desnutrição neonatal sobre o recrutamento celular e a atividade oxidante--antioxidante de macrófagos em ratos adultos endotoxêmicos. Rev Nutr. 2008; 21(6):683-94. doi: 10.1590/S1415-52732008000600007
https://doi.org/10.1590/S1415-5273200800...
, but they used a regional basic diet low in all constituents. Costa et al. 55. Costa TB, Morais NG, Almeida TM, Severo MS, Castro CMMB. Early malnutrition and production of IFN-?, IL-12 and IL-10 by macrophages/lymphocytes: In vitro study of cell infection by methicillin-sensitive and methicillin-resistant Staphylococcus aureus. Rev Nutr. 2012; 25(5):607-19. doi: 10.1590/S1415-52732012000500006
https://doi.org/10.1590/S1415-5273201200...
used an 8% casein diet to induce malnutrition and also found that weight gain decreased after the fourth day of life.

Nutritional insults in the neonatal period seem to interfere with the programming of macrophage functional mechanisms, causing lasting changes detectable in adulthood, even after a long nutritional recovery33. Melo JF, Costa TC, Lima TDC, Chaves MEC, Vayssade LM, Nagel MD, et al. Long-term effects of a neonatal low-protein diet in rats on the number of macrophages in culture and the expression/production of fusion proteins. Eur J Nutr. 2013; 52(5):1475-82. doi: 10.1007/s00394-012-0453-y
https://doi.org/10.1007/s00394-012-0453-...
. Prestes-Carneiro et al. 2020. Prestes-Carneiro LE, Laraya RD, Silva PRC, Moliterno RA, Felipe I, Mathias PC. Long-term effect of early protein malnutrition on growth curve hematological parameters and macrophage function of rats. J Nutr Scien Vitaminol. 2006; 52(6):414-20. doi: 10.3177/jnsv.52.414
https://doi.org/10.3177/jnsv.52.414...
found that malnutrition from the first to twelfth day of lactation compromised the microbicidal response, represented by lower rate of phagocytosis. Other researchers have reported a deficit in the production of nitric oxide44. Melo JF, Macedo EMC, Silva RPP, Viana MT, Silva WTF, Castro CMMB. Efeito da desnutrição neonatal sobre o recrutamento celular e a atividade oxidante--antioxidante de macrófagos em ratos adultos endotoxêmicos. Rev Nutr. 2008; 21(6):683-94. doi: 10.1590/S1415-52732008000600007
https://doi.org/10.1590/S1415-5273200800...
, 2121. Anstead GM, Chandrasekar B, Zhao W, Yang J, Perez LE, Melby PC. Malnutrition alters the innate immune response and increases early visceralization following Leishmania donovani infection. Infect Immun. 2001; 69:4709-18. doi: 10.1128/IAI.69.8.4 709-4718.2001
https://doi.org/10.1128/IAI.69.8.4709-47...
. Dong et al. 2222. Dong W, Selgrade MJK, Gilmour MI, Lange RW, Park P, Luster MI, et al. Altered alveolar macrophage function in calorie-restricted rats. Am J Respir Cell Mol Biol. 1998; 19(1):462-9. doi: 10.1165/ajrcmb. 19.3.3114
https://doi.org/10.1165/ajrcmb19.3.3114...
reported lower production of free radicals by alveolar macrophages after in vitro stimulation with LPS.

In the present study, the low-protein diet did not change alveolar macrophage adherence regardless of pathogenic stimulus, suggesting that this initial step of the macrophages' immune response may not be impaired by neonatal malnutrition caused by an 8% casein diet. Chandra22. Chandra RK. Nutrition and the immune system from birth to old age. Eur J Clin Nutr. 2002; 56(3):73-6. doi: 10.1038/sj.ejcn.1601492
https://doi.org/10.1038/sj.ejcn.1601492...
noticed that malnutrition changes different stages of activated neutrophil and macrophage phagocytosis. Thus, malnutrition may change mechanisms that rely on macrophage activation not necessarily before activation.

According to this premise, the study demonstrated that malnutrition during lactation reduced macrophage phagocytosis in both systems. According to Prestes-Carneiro et al. 2020. Prestes-Carneiro LE, Laraya RD, Silva PRC, Moliterno RA, Felipe I, Mathias PC. Long-term effect of early protein malnutrition on growth curve hematological parameters and macrophage function of rats. J Nutr Scien Vitaminol. 2006; 52(6):414-20. doi: 10.3177/jnsv.52.414
https://doi.org/10.3177/jnsv.52.414...
in the case of inflammatory stimuli, macrophages from malnourished animals do not respond with the same intensity as macrophages from nourished animals, which allows the development of inflammation and/or infection. Other studies have also reported low phagocytic capacity in animals subjected to malnutrition, whether neonatal or not44. Melo JF, Macedo EMC, Silva RPP, Viana MT, Silva WTF, Castro CMMB. Efeito da desnutrição neonatal sobre o recrutamento celular e a atividade oxidante--antioxidante de macrófagos em ratos adultos endotoxêmicos. Rev Nutr. 2008; 21(6):683-94. doi: 10.1590/S1415-52732008000600007
https://doi.org/10.1590/S1415-5273200800...
, 2222. Dong W, Selgrade MJK, Gilmour MI, Lange RW, Park P, Luster MI, et al. Altered alveolar macrophage function in calorie-restricted rats. Am J Respir Cell Mol Biol. 1998; 19(1):462-9. doi: 10.1165/ajrcmb. 19.3.3114
https://doi.org/10.1165/ajrcmb19.3.3114...
.

Regarding oxidant activity, alveolar macrophages from neonatally malnourished rats produced less superoxide, both under normal conditions and under bacterial stimuli. Corroborating this finding, Kawakami et al. 23 23. Kawakami K, Kadota J, Lida K, Shirai R, Abe K, Kohno S. Reduced immune function and malnutrition in the elderly. Tohoku J Exp Med. 1999; 187(2):157-71.found that phagocytes' antimicrobial systems are potentially affected by malnutrition. Prestes-Carneiro et al. 2020. Prestes-Carneiro LE, Laraya RD, Silva PRC, Moliterno RA, Felipe I, Mathias PC. Long-term effect of early protein malnutrition on growth curve hematological parameters and macrophage function of rats. J Nutr Scien Vitaminol. 2006; 52(6):414-20. doi: 10.3177/jnsv.52.414
https://doi.org/10.3177/jnsv.52.414...
also found that superoxide production decreases during severe protein malnutrition.

In the present study, nitric oxide production was analyzed every two hours for a total of 24 hours of incubation. Both groups produced more than the PC, peaking at 22 hours, but malnutrition decreased production. Corroborating this result, Melo et al. 44. Melo JF, Macedo EMC, Silva RPP, Viana MT, Silva WTF, Castro CMMB. Efeito da desnutrição neonatal sobre o recrutamento celular e a atividade oxidante--antioxidante de macrófagos em ratos adultos endotoxêmicos. Rev Nutr. 2008; 21(6):683-94. doi: 10.1590/S1415-52732008000600007
https://doi.org/10.1590/S1415-5273200800...
found that alveolar and peritoneal macrophages produced less nitric oxide after 24 hours of incubation with LPS in rats submitted to early malnutrition. Ferreira-Silva et al. 2424. Ferreira-Silva WT, Galvão BA, Ferraz Pereira KN, Castro CMMB, Manhaes-de-Castro R. Perinatal malnutrition programs sustained alterations in nitric oxide release by activated macrophages in response to fluoxetine in adult rats. Neuroimmunomodulation. 2009; 16(4):219-27. doi: 10.1159/000212382
https://doi.org/10.1159/000212382...
also found that nitrite concentration decreased in cell culture supernatant during nitric oxide production in the undernourished group after LPS stimulation. These data indicate that neonatal malnutrition induces changes in the macrophages, with significant repercussions during adulthood99. Mandell GL. Uptake, transport, delivery and intracellular activity of antimicrobial agents. Pharmacother. 2005; 25(12):130S-3S..

Pumerantz et al. 2525. Pumerantz A, Muppidi K, Agnihotri S, Guerrac C, Venketaraman V, Wangb J, et al. Preparation of liposomal vancomycin and intracellular killing of Meticillin-Resistant Staphylococcus Aureus (MRSA). Internat J Antimicrob Ag. 2010; 37(2):140-4. doi: 10.1016/j.ijantimicag.2010.10.011
https://doi.org/10.1016/j.ijantimicag.20...
stated that the nitric oxide produced by alveolar macrophages plays an important microbicide role against Staphylococcus aureus. By comparing the nitric oxide release in the MSSA and MRSA systems of nourished and malnourished groups, the malnourished groups presented the lowest production. Low synthesis of this free radical may allow resistant bacteria to proliferate inside phagocytes because this important defense mechanism is compromised1010. Morais NG, Costa TB, Almeida TM, Severo MS, Castro CMMB. Parâmetros imunológicos de macrófagos frente à infecção por Staphylococcus aureus meticilina sensível/resistente. J Bras Patol Med Lab. 2013; 49(2):84-90. doi: 10.1590/S1676-24 442013000200002
https://doi.org/10.1590/S1676-2444201300...
.

According to Richardson et al. 2626. Richardson AR, Libby SJ, Fang FC. A nitric oxide-inducible lactate dehydrogenase Enables Staphylococcus aureus to resist innate immunity. Scien. 2008; 319(5870):1672-6. doi: 10.1126/science. 1155207
https://doi.org/10.1126/science.1155207...
, S. aureus can evade multiple components of the innate immune response, including the microbicidal action of nitric oxide. These authors found that S. aureus can adapt metabolically to nitrosative stress because it has an inducible NO-L-lactate dehydrogenase enzyme. The production of NO-L-lactate dehydrogenase enables S. aureus to keep homeostasis during nitrosative stress, and antibiotic resistance does not seem to interfere on this mechanism.

Based on analysis of IL-1β production, the nourished and malnourished groups differed only on the testing systems. High IL-1β production was detected in the MSSA system of the nourished group, but in the MRSA system, it was higher in the malnourished groups. IL-1β is a potent endogenous pyrogen (a fever inducer), and a potent stimulator of leukocyte migration into tissues and cytokine and chemokine expression2727. Latz E. The inflammasomes: Mechanisms of activation and function.Curr Opin Immunol. 2010; 22(1):28-33. doi: 10.1016/j.coi.2009.12.004
https://doi.org/10.1016/j.coi.2009.12.00...
. IL-1β is an important mediator for defense against Staphylococcus aureus. In S. aureus infection, the production of IL-1β acts in the recruitment of neutrophils and the subsequent degradation of the bacterial cell wall by lysozyme enzyme. However, S. aureus has an O-acetyltransferase enzyme that transforms the cell wall resistant to the action of lysozyme and thus escapes the microbicidal response2828. Lalor SJ, Dugan LS, Sutton CE, Basdeo SA, Fletcher JM, Mills KHG. IL-18 promote IL-17 production by gd and caspase-1-processed cytokines IL-1b and CD4 T cells that mediate autoimmunity. J Immunol. 2011; 6(1):55-63. doi: 10.4049/jimmunol.100 3597
https://doi.org/10.4049/jimmunol.1003597...
. These findings justify the high MRSA-related mortality rates.

IL-18 production was higher in the positive than negative control. When analyzing the testing systems of the nourished and malnourished groups, the production in the malnourished groups was small and even smaller in the MRSA system. IL-18 induces the production of IFN-y (interferon-gamma) by cells of the immune system. This cytokine is important for the activation of macrophages, T lymphocytes, and other cells2828. Lalor SJ, Dugan LS, Sutton CE, Basdeo SA, Fletcher JM, Mills KHG. IL-18 promote IL-17 production by gd and caspase-1-processed cytokines IL-1b and CD4 T cells that mediate autoimmunity. J Immunol. 2011; 6(1):55-63. doi: 10.4049/jimmunol.100 3597
https://doi.org/10.4049/jimmunol.1003597...
. In MRSA infections of malnourished animals, the pro-inflammatory profile (Th1) may be compromised, favoring the persistence of the bacteria in the host organism.

When comparing macrophage viability in the PC, MSSA, and MRSA systems, viability decreased intensely after infection with S. aureus. This finding was more evident in the malnourished group infected by MRSA. This indicates that macrophage vulnerability is greater during MRSA infection, especially in immunocompromised individuals.

Protein-calorie deficiencies may induce irreversible cell damage that triggers the mechanism of programmed cell death2020. Prestes-Carneiro LE, Laraya RD, Silva PRC, Moliterno RA, Felipe I, Mathias PC. Long-term effect of early protein malnutrition on growth curve hematological parameters and macrophage function of rats. J Nutr Scien Vitaminol. 2006; 52(6):414-20. doi: 10.3177/jnsv.52.414
https://doi.org/10.3177/jnsv.52.414...
. Ferreira-Silva et al. 2424. Ferreira-Silva WT, Galvão BA, Ferraz Pereira KN, Castro CMMB, Manhaes-de-Castro R. Perinatal malnutrition programs sustained alterations in nitric oxide release by activated macrophages in response to fluoxetine in adult rats. Neuroimmunomodulation. 2009; 16(4):219-27. doi: 10.1159/000212382
https://doi.org/10.1159/000212382...
found a reduction in the viability of alveolar macrophages after perinatal malnutrition. Corroborating these authors, Rivadeneira et al. 2929. Rivadeneira DE, Grobmyer SR, Naama HA, Mackrell PJ, Mestre JR, Stapleton PP, et al. Malnutrition-induced macrophage apoptosis. Surgery. 2001; 129(5):617-25. doi: 10.1067/msy.2001.112963
https://doi.org/10.1067/msy.2001.112963...
found that malnutrition is associated with increased apoptosis. Apoptosis can be triggered by external stimuli or internal stimuli that result in mitochondrial dysfunction, DNA damage, and alteration of nutritional and growth factors. These different routes induce activation of caspases, which generate the cleavage of structural proteins, impairing cytoskeleton integrity, resulting in cell death2929. Rivadeneira DE, Grobmyer SR, Naama HA, Mackrell PJ, Mestre JR, Stapleton PP, et al. Malnutrition-induced macrophage apoptosis. Surgery. 2001; 129(5):617-25. doi: 10.1067/msy.2001.112963
https://doi.org/10.1067/msy.2001.112963...
.

Staphylococcus aureus is able to produce a variety of potent cytotoxins, allowing the bacteria to resist microbicidal response. Leucocidin is a toxin associated with new methicillin-resistant Staphylococcus aureus strains that destroys leukocytes by forming pores in the cell membrane3030. Nizet V. Understanding how leading bacterial pathogens subvert innate immunity to reveal novel therapeutic targets. J Allergy Clin Immunol. 2007; 120(1):13 22. doi: 10.1016/j.jaci.2007.06.005
https://doi.org/10.1016/j.jaci.2007.06.0...
. Thus, we suggest that S. aureus infection induced phagocyte death by triggering cell lysis, and that neonatal undernutrition further promoted this effect. The study results may explain the high morbidity and mortality rates associated with MRSA infection in immunocompromised individuals.

CONCLUSION

The study neonatal malnutrition model compromised some functional parameters of innate immunity, such as rate of phagocytosis and production of nitric oxide, superoxide anion, and IL-18. Phagocytosis and the production of these inflammatory mediators are critical for the effective destruction of invading microorganisms. Adherence rate and production of IL-1β were not affected, but neonatal nutrition does impact the programming of macrophage microbicidal mechanisms. Methicillin-sensitivity in Staphylococcus aureus strains seems to influence their ability to evade the microbicidal response, decreasing immune defense. Interaction between neonatal malnutrition and MRSA infection increased phagocyte susceptibility, which may allow severe and fatal infections. However, many gaps remain to be filled regarding the structure and performance of immune defense components during infections, such as those caused by Staphylococcus aureus in adults who have endured environmental insults. Thus, it is important to conduct studies using more sensitive and specific methods, such as biological molecular analyses. These may provide better data on this topic and contribute to the clarification of the morphological changes that occur in early life and the impact of such changes on the microbicidal response of phagocytes and on the emergence and evolution of infectious diseases.

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Publication Dates

  • Publication in this collection
    Sep-Oct 2014

History

  • Received
    29 Jan 2014
  • Reviewed
    15 July 2014
  • Accepted
    05 Aug 2014
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