Beneficial effects of a low-protein diet on host resistance to Paracoccidioides brasiliensis in mice

Nutrition 25 (2009) 954–963

Basic nutritional investigation

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Beneficial effects of a low-protein diet on host resistance to Paracoccidioides brasiliensis in mice Motoko Oarada, Ph.Da,*, Katsuhiko Kamei, M.D., Ph.D.a, Tohru Gonoi, Ph.D.a, Tsuyoshi Tsuzuki, Ph.D.b, Takahito Toyotome, Ph.D.a, Katsuya Hirasaka, Ph.D.c, Takeshi Nikawa, M.D., Ph.D.c, Ayaka Sato B.Sc.a, and Nobuyuki Kurita, Ph.D.a b

a Medical Mycology Research Center, Chiba University, Chiba, Japan Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan c Department of Nutrition, Tokushima University School of Medicine, Tokushima, Japan

Manuscript received August 23, 2008; accepted February 24, 2009

Abstract

Objective: Although protein malnutrition impairs immune functions, several studies have recently shown that protein restriction without malnutrition is beneficial to host defenses against invading pathogens and cancer. In an effort to establish the optimum diet for host resistance, we investigated the effect of different dietary protein levels on host resistance to Paracoccidioides brasiliensis. Methods: Mice were fasted for 2 days and then infected with P. brasiliensis. Immediately after challenge with this fungus, mice were refed on diets with three different levels (0%, 1.5%, or 20%) of casein. On days 0–7 after infection, antifungal activity and levels of proinflammatory mediators in the spleen and liver were measured. Results: Mice refed on the 1.5% casein diet showed higher antifungal activity in the spleen and liver compared with mice on the 20% casein diet. The antifungal activity in the spleens of mice refed on the 0% casein diet was intermediate between the antifungal activities of those refed the 1.5% and 20% casein diets. After infection, increases in spleen and liver levels of interleukin-6 and interferon-g, liver mRNA levels of antimicrobial proteins (myeloperoxidase, cathepsin-G, and elastase-2), and liver mRNA levels of proinflammatory mediators (interleukin-18, chemokine C-X-C motif ligand 10, nuclear factor-kB, inducible nitric oxide synthase, and granulocyte–macrophage colony-stimulating factor) were less profound in mice on the 1.5% or 0% casein diet compared with mice refed the 20% casein diet. Conclusion: The present results suggest that protein restriction without malnutrition could be beneficial to host resistance to P. brasiliensis. Ó 2009 Elsevier Inc. All rights reserved.

Keywords:

Antifungal activity; Interleukin-6; Infection; Interferon-g; Low-protein diet; Proinflammatory mediator; Protein restriction without malnutrition

Introduction It is generally accepted that the normal dietary protein level (as casein) is around 20%, because the optimum dietary casein level for growth of rodents has been shown to be 18% to 25% (w/w) [1]. In protein malnutrition, which is characterized by a reduction in the levels of blood total protein and albumin [2,3], a variety of deleterious physiologic effects This study was supported in part by the Kieikai Research Foundation. *Corresponding author. Tel.: þ81-43-226-2787; fax: þ81-43-226-2486. E-mail address: [email protected] (M. Oarada). 0899-9007/09/$ – see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.02.004

such as reduction in growth rate [4] and impairment of immunity including compromised cytokine responses, thymic atrophy, and reduced immunoglobulin A and G production have been observed [5,6]. Conversely, it has been recently reported that dietary protein restriction without malnutrition could have beneficial effects on host defenses against invading pathogens and cancer. Hu et al. [7] reported that a low-protein diet (6% casein) reduces expression of hepatitis B virus (HBV) in HBV transgenic mice compared with a 22% casein diet. It has been shown also that complete protection against malaria was achieved by consumption of lowprotein diets (0%, 5%, and 12.5% protein) for 1 wk before

M. Oarada et al. / Nutrition 25 (2009) 954–963

infection compared with consumption of a 25% protein diet [8]. In addition, a 5% protein diet exerts a protective effect against tumor metastasis compared with a 25% protein diet [9]. However, information concerning the effect of lowprotein diets on host resistance and immune functions is still fragmentary. In the present study, we investigated the effects of different dietary protein levels on host resistance to Paracoccidioides brasiliensis and the production of proinflammatory mediators. We report that a 1.5% casein diet enhanced host resistance to P. brasiliensis and reduced the production of proinflammatory mediators compared with a 20% casein diet. Materials and methods Diets Casein, a-corn starch, sucrose, cellulose powder, AIN-76 mineral mixture [10], AIN-76 vitamin mixture [10], and choline bitartrate were purchased from Oriental Yeast (Tokyo, Japan). DL-methionine was obtained from Wako Pure Chemical Industries (Osaka, Japan). Soybean oil was supplied by NOF (Tokyo, Japan). Using food-grade ingredients, purified rodent powder diets were prepared in our laboratory. Composition of the test diets is presented in Table 1. The three dietary groups examined in our analyses could be differentiated by their casein weight percentage: 0%, 1.5%, or 20%. Casein was exchanged isoenergetically with a-corn starch. Fungi Paracoccidioides brasiliensis isolate Pb-18, isolated from a Brazilian patient with paracoccidioidomycosis, was used to infect the mice. The fungal cells used in the experiments were newly derived from the mycelial form of this microbe and were subcultured twice at 35  C at 4-d intervals on 1% glucose-supplemented brain–heart infusion (Difco Laboratories, Detroit, MI, USA) agar slants. Fresh growth (4 d) of the fungus was collected in 0.9% sterile saline with mesh to Table 1 Composition of test diets Composition (g/kg)

Casein DL-Methionine a-Corn starch Sucrose Soybean oil* Mineral mixturey Vitamin mixturey Choline bitartrate Cellulose

Casein in diet 20%

1.5%

0%

200 3 550 100 50 35 10 2 50

15 0.2 737.8 100 50 35 10 2 50

0 0 753 100 50 35 10 2 50

* Fatty acid composition (grams per 100 g of fatty acid): 16:0, 17.4; 16:1(u-9), 0.1; 18:0, 5.7; 18:1(u-9), 22.3; 18:1(u-7), 1.2; 18:2(u-6), 46.9; 18:3(u-3), 6.1; 20:1(u-9), 0.2; 22:5(u-3), 0.1. y AIN-76 mineral and vitamin mixtures [11].

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eliminate cell clumps. After washing once, the fungal cells were counted using a hemacytometer. More than 97% of the fungal cells prepared in this manner were viable and resuspended at the indicated densities in saline before infecting the mice. Animals and experimental design Specific-pathogen–free, 5-wk-old female BALB/c mice were obtained from Charles River Japan (Atsugi, Japan). The animals were maintained on a commercial laboratory chow (Oriental Yeast) and water ad libitum. Approximately 100 g of the non-purified diet contained 23.6 g of protein, 5.3 g of fat, 6.1 g of ash, 2.9 g of fiber, and 54.4 g of nitrogen-free extracts. After an acclimatization period (5 d), mice were fed the test diet containing 20% casein and water ad libitum. One week after commencing the 20% casein diet, the mice were deprived of food for a period of 48 h. During this period of food deprivation, mice were allowed free access to water. After the 48-h food-deprivation period, the mice were infected using a single intravenous injection of a 200-mL fungal cell suspension (0.9 3 107 cells/mL) through the lateral tail vein. After infection, mice were immediately assigned to a test diet containing 0%, 1.5%, or 20% casein. These diets were consumed ad libitum for 7 d. Infected mice consuming the test diets were sacrificed by decapitation on days 0–7 after infection and their blood, spleens, and livers were harvested. All animal housing, handling, and sample collection procedures described conformed to the policies and recommendations of the laboratory animal care advisory committee of Chiba University (Chiba, Japan). Measurement of serum urea nitrogen, total protein, and albumin levels Blood was collected and allowed to clot for 1 h at room temperature. Serum was then separated by centrifugation at 3000 rpm for 20 min at 4  C. Serum urea nitrogen, total protein, and albumin levels were measured using a urea nitrogen B-test (Wako Pure Chemical Industries), a BCA Protein Assay Kit (PIERCE, Rockford, IL), and an A/G B-test (Wako Pure Chemical Industries), respectively. All assays were performed in duplicate and data averages were statistically analyzed. Counts of viable fungal cells from organs Spleens and livers were aseptically removed from the subject mice and homogenized in a glass homogenizer with 4 and 19 mL of saline, respectively. Samples were diluted with saline. One milliliter of each diluted homogenate was then plated on brain–heart infusion agar supplemented with 50 mmol/L of ethylenediaminetetra-acetic acid, 20 mL/L of horse serum (GIBCO Laboratories, Grand Island, NY, USA), and 150 mmol/L of chloramphenicol (Wako Pure Chemical Industries). The agar plates were incubated in a humidified atmosphere at 35  C and the colony-forming units

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(CFUs) of P. brasiliensis were counted after 21 d of incubation. Measurement of interleukin-6 and interferon-g levels Mouse spleens and livers were homogenized in ice-cold phosphate buffered saline (10%, v/v) with a glass handheld tissue homogenizer and centrifuged at 10 000 rpm for 30 min at 4  C to pellet the debris. The levels of interleukin (IL)-6 and interferon (IFN)-g were determined in sample supernatants using enzyme-linked immunosorbent assay kits purchased from Endogen (Rockford, IL, USA). All assays were performed according to the manufacturer’s instructions. RNA extraction and quantitative real-time reverse transcriptase polymerase chain reaction Total RNA was isolated from mouse spleens and livers with an RNAeasy extraction kit according to the manufacturer’s instructions (Qiagen, Santa Clarita, CA, USA). Complementary DNAs were synthesized from 2 mg of individual total RNA extracts using oligo-dT primers and a reverse transcription system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Real-time quantitative reverse transcriptase polymerase chain reaction analyses were performed in a final volume of 20 mL using a SYBR Green polymerase chain reaction master mix (Applied Biosystems, Foster City, CA, US) containing 300-nM primers and 10 ng of reverse transcribed total RNA. The resulting amplified products were analyzed in real time using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems). The primer sequences used were as follows (product size and GenBank accession number are indicated in parentheses): myeloperoxidase (145 bp, BC053912), forward 50 -CCGCCTGAACAATCAGTACC-30 , reverse 50 -A TTCAGTTTGGCTGGAGTGG-30 ; cathepsin-G (186 bp, NM_007800), forward 50 -AGGACCCAGCAACTCATCA C-30 , reverse 50 -AGCCACTGTGCACAAATCC-30 ; elastase2 (151 bp, NM_015779), forward 50 -TGCTACTGGCATTG TTCCTG-30 , reverse 50 -ACGAAGTTCCTGGCAATGAG-30 ; IL-18 (193 bp, NM_008360), forward 50 -GGCCGACTTC ACTGTACAAC-30 , reverse 50 -CCTTCACAGAGAGGGTCA CAG-30 ; chemokine C-X-C motif ligand 10 (CXCL10; 152 bp, NM_021274), forward 50 -GGTCTGAGTGGGACTCAAG G-30 , reverse 50 -GTGGCAATGATCTCAACACG-30 ; inducible nitric oxide synthase (iNOS; 144 bp, NM_010927), forward 50 -CCCGCTACTACTCCATCAGC-30 , reverse 50 -GCTTCA GGTTCCTGATCCAA-30 ; granulocyte–macrophage colonystimulating factor (GM-CSF; 105 bp, BC116880), forward 50 -T GGTCTACAGCCTCTCAGCA-30 , reverse 50 -GCATGTCA TCCAGGAGGTTC-30 ; nuclear factor (NF)-kB (107 bp, AY521463), forward 50 -AGGCCATTGAAGTGATCCAG-30 , reverse 50 -CGTGGAGGAAGACGAGAGAG-30 ; glyceraldehyde-3-phosphate dehydrogenase (177 bp, M32599), forward 50 -TGCACCACCAACTGCTTAG-30 , reverse 50 -GGATGCA GGGATGATGTTC-30 . The relative expression levels of the

target gene products were calculated with the comparative threshold cycle method using glyceraldehyde-3-phosphate dehydrogenase as the normalization control. Statistical analysis All data were expressed as mean 6 standard deviation (SD) for n observations. Comparisons among the three dietary groups for each day and variations of CFU count within dietary groups over time were evaluated using SPSS software (SPSS Inc., Japan, Tokyo) and analyis of variance. Tukey’s B test was used for any post hoc analysis. Data comparisons between day 0 and days 1, 3, 5, or 7 after infection were made using Levene’s test and an unpaired Student’s t test with SPSS. Results Food intake and total body, spleen, and liver weights Daily food intakes of mice refed the 20% casein diet were 4.3 6 0.1, 3.1 6 0.7, 1.9 6 0.3, 3.4 6 0.6, 3.4 6 0.6, 3.6 6 0.1, and 2.2 6 0.1 g/mouse on days 1, 2, 3, 4, 5, 6, and 7 after infection, respectively. A similar pattern of food intake was also observed in mice refed the 1.5% or 0% casein diets (data not shown); there was no significant difference in food intake between the different dietary groups. Two-day starvation resulted in decreases in body weight from 19.8 6 0.8 g (mean 6 SD, before starvation) to 16.9 6 0.6 g (P < 0.05, n ¼ 10/group). At days 3, 5, and 7 after infection, the mean body weight of mice refed the 20% casein diet was significantly higher than that of mice refed the 1.5% or 0% casein diets (Fig. 1A). Two-day starvation reduced the spleen weight (prestarved versus starved, 106 6 17 versus 76 6 10 mg, P < 0.05, n ¼ 10/group). At days 3, 5, and 7 after infection, mice refed the 20% casein diet showed a significantly greater increase in spleen weight compared with mice refed the 1.5% or 0% casein diet (Fig. 1B). Two-day starvation also decreased the mean liver weight (prestarved versus starved, 1.08 6 0.03 versus 0.86 6 0.06 g, P < 0.05, n ¼ 10/group). At days 3, 5, and 7 after infection, mice refed the 20% casein diet had significantly higher liver weights compared with mice refed the 1.5% or 0% casein diet (Fig. 1C). Serum urea nitrogen, total protein, and albumin levels Two-day starvations had no significant effects on serum urea nitrogen levels (prestarved versus starved, 18.00 6 1.00 versus 16.20 6 2.84 mg/dL, n ¼ 5/group). It is well known that dietary protein restriction decreases serum urea nitrogen levels [11,12]. In mice refed the 1.5% and 0% casein diets, serum urea nitrogen levels decreased to 7.6 6 1.0 and 6.3 6 1.1 mg/dL by 3 h after infection, respectively, and stayed at lower levels until day 7 (Fig. 2A). In contrast, in mice refed the 20% casein diet, serum urea nitrogen levels increased by 3 h after infection and then returned to the normal level (day 0 in

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Fig. 1. Body (A), spleen (B), and liver (C) weights in Paracoccidioides brasiliensis–infected mice refed diets with three levels (0%, 1.5%, or 20%) of casein. Each value represents the mean 6 SD (n ¼ 5/test group at each day). Day 0, before infection/refeeding. Values highlighted with different superscripts (a–c) for the same day are significantly different (P < 0.05). Values highlighted with asterisks are significantly different from day 0 (P < 0.05).

A

B

C

Fig. 2. Serum urea nitrogen (A), total protein (B), and albumin (C) levels in Paracoccidioides brasiliensis–infected mice refed diets with three levels (0%, 1.5%, or 20%) of casein. Each value represents the mean 6 SD (n ¼ 5/test group at each day). Day 0, before infection/refeeding. Day 0 (3 h), 3 h after infection/refeeding. Values highlighted with different superscripts (a–c) for the same day are significantly different (P < 0.05). Values highlighted with asterisks are significantly different from day 0 (P < 0.05).

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Table 2 Recovery of Paracoccidioides brasiliensis from spleens of mice refed diets with three levels (0%, 1.5%, and 20%) of casein* Casein in diet

Colony-forming units

20% 1.5% 0%

0y

1

3

5

7

5088vw 6 660 4938v 6 1054 5669v 6 1278

3964w 6 516 3415w 6 277 3350w 6 979

6602a,v 6 2292 3465b,w 6 626 5084ab,v 6 1605

3468a,wx 6 249 1461b,x 6 464 2048b,wx 6 447

1413a,x 6 336 565b,x 6 105 906ab,x 6 417

* Data are means 6 SDs (n ¼ 5/group on each day). Mean values with different superscript letters (a,b) for the same day are significantly different (P < 0.05). Mean values with different superscript letters (v–x) within the indicated dietary group are significantly different over time (P < 0.05). y Time after infection (days). Day 0, 3 h after infection/refeeding.

Fig. 2A) by day 5. Two-day starvations did not significantly affect serum total protein levels (prestarved versus starved, 5.12 6 0.45 versus 5.52 6 0.39 g/dL, n ¼ 10/group). In mice refed the 1.5% or 0% casein diet, serum total protein levels were maintained at the normal level until day 7 after infection (Fig. 2B). The 2-d starvation conditions also had no significant effects on serum albumin levels (prestarved versus starved, 3.11 6 0.11 versus 3.08 6 0.10 g/dL, n ¼ 5/group). In mice refed the 1.5% and 0% casein diets, serum albumin levels only slightly decreased from normal levels by day 5 after infection (Fig. 2C). There was no significant difference between mice refed the 20% casein diet and the 1.5% or 0% casein diet in serum albumin levels at days 1 and 5 after infection, and mice refed the 1.5% or 0% casein diet showed slightly lower levels of serum albumin compared with mice refed the 20% casein diet at days 3 and 7. Antifungal resistance The CFU counts obtained from the spleens were not significantly different among mice refed on the three levels (0%, 1.5%, or 20%) of casein at day 0 after infection (Table 2). However, at days 3, 5, and 7 after infection, the mean spleen CFU count from mice refed a diet containing 1.5% casein was approximately 50% of that recorded for mice refed on the 20% casein diet. The mean spleen CFU count for mice refed on the 0% casein diet was two-thirds of the mean found in mice refed the 20% casein diet at day 5 after infection. The liver CFU counts from mice refed the 0% casein diet were 1.3- and 1.4-fold higher than those from mice refed the 20% casein diet at days 0 and 1 after infection, respectively

(Table 3). At days 0, 1, and 3 after infection, there was no significant difference found in the liver CFU counts between mice refed the 20% and 1.5% casein diets. However, at days 5 and 7 after infection, the liver CFU counts from mice refed on 1.5% casein diet were approximately 50% of those obtained from mice refed the 20% casein diet. Mice refed on 0% casein diet showed lower liver CFU counts at day 5 compared with mice refed the 20% casein diet. IL-6 and IFN-g levels in spleen and liver Cellular, but not humoral, immunity has been shown to be decisive in host resistance to P. brasiliensis in the murine system [13], and IFN-g and IL-6 have been suggested to play a pivotal defensive role in this response [14,15]. Two-day starvation had no significant effects on IL-6 and IFN-g levels in the spleen (data not shown). At days 3 and 5 after infection, mice refed the 20% casein diet showed higher spleen IL-6 levels compared with mice refed the 1.5% or 0% casein diet (Fig. 3A). Spleen IFN-g levels in mice refed the 20% casein diet were 1.8- and 1.5-fold higher at day 3 than in mice refed the 1.5% and 0% casein diets, respectively (Fig. 3B). Two-day starvation also had no significant effects on mouse liver IL-6 levels (data not shown), but resulted in an increase in the liver IFN-g concentration from 16.76 6 0.86 (prestarved) to 21.79 6 1.40 ng/g of tissue (P < 0.05, n ¼ 5/group). At day 3 after infection, mice refed the 20% casein diet exhibited 2.0- and 2.4-fold higher liver IL-6 levels compared with mice refed the 1.5% and 0% casein diets, respectively (Fig. 3C). At day 3 after infection, the liver IFN-g levels of mice refed the 20% casein diet were

Table 3 Recovery of Paracoccidioides brasiliensis from livers of mice refed three levels (0%, 1.5%, and 20%) of casein diet* Casein in diet

Colony-forming units 0y

20% 1.5% 0%

1 b,w

34 234 6 4400 38 133ab,w 6 3529 45 890a,w 6 5907

3 b,x

23 023 6 2536 25 567b,x 6 1573 31 832a,wx 6 3489

5 v

65 057 6 4288 50 855v 6 5515 70 155v 6 27871

7 a,w

33 251 6 5451 15 147c,y 6 1674 22 249b,xy 6 2646

11 504a,y 6 3388 5646b,y 6 1916 8995ab,y 6 2270

* Data are means 6 SDs (n ¼ 5/group on each day). Mean values with different superscript letters (a–c) for the same day are significantly different (P < 0.05). Mean values with different superscript letters (v–y) within the indicated dietary group are significantly different over time (P < 0.05). y Time after infection (days). Day 0, 3 h after infection/refeeding.

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Fig. 3. Levels of IL-6 in the spleen (A), IFN-g in the spleen (B), IL-6 in the liver (C), and IFN-g in the liver (D) in Paracoccidioides brasiliensis–infected mice refed diets with three levels (0%, 1.5%, or 20%) of casein. Each value represents the mean 6 SD (n ¼ 5/test group at each day). Day 0, before infection/refeeding. Values highlighted with different superscripts (a,b) for the same day are significantly different (P < 0.05). Values highlighted with asterisks are significantly different from day 0 (P < 0.05). IL-6, interleukin-6; IFN-g, interferon-g.

1.4-fold higher than in mice refed the 1.5% casein diet (Fig. 3D).

Expression of specific genes in the liver The 2-d starvation conditions had no significant effects on the mouse liver mRNA levels of myeloperoxidase, cathepsin-G, elastase-2, CXCL10, NF-kB, iNOS, or GMCSF (data not shown), but resulted in a reduction in liver IL-18 transcript levels (prestarved versus starved, 0.80 6 0.08 versus 0.59 6 0.06, P < 0.05, n ¼ 5/group). At day 5 after infection, liver transcript levels of myeloperoxidase, cathepsin-G, and elastase-2 were 6.2- to 8.1-fold higher in mice refed the 20% casein diet compared with mice refed the 1.5% or 0% casein diet (Fig. 4A–C). Mice refed the 20% casein diet had higher liver IL-18 mRNA expression at days 1, 3, and 5 after infection than mice refed the 1.5% or 0% casein diet (Fig. 4D). In mice refed the 20% casein diet, liver CXCL10 mRNA levels were 1.8fold higher than in mice refed the 1.5% casein diet at day 3 after infection (Fig. 4E). Mice refed the 20% casein diet showed higher liver expression of NF-kB transcripts compared with mice refed the 1.5% casein diet at days 3 and 5 after infection (Fig. 4F). The mRNA levels of iNOS and GM-CSF in the liver were 1.9- to 2.1-fold higher in mice refed the 20% casein diet than in mice refed the 1.5% or 0% casein diet at day 5 after infection (Fig. 4 G,H).

Expression of specific genes in the spleen Two-day starvation had no significant effect on spleen mRNA levels of myeloperoxidase, cathepsin-G, elastase-2, and iNOS (data not shown). Mice refed the 20% casein diet showed 2.8- to 8.1-fold higher mRNA expression of myeloperoxidase, cathepsin-G, and elastase-2 in the spleen compared with mice refed the 1.5% or 0% casein diet at day 5 after infection (Fig. 5A–C). Spleen iNOS mRNA levels in mice refed the 20% casein diet were 1.7- and 2.3-fold higher than those in mice refed 1.5% casein diet at days 3 and 5 after infection, respectively (Fig. 5D). There was no significant difference in spleen mRNA levels of IL-18, CXCL10, and GM-CSF among the different casein dietary groups (data not shown). Discussion There is ample evidence in the literature to indicate that protein malnutrition has deleterious effects on immune function, which predisposes individuals to an increased risk of infection [16,17]. In contrast, in the present study, a low-protein (1.5% casein) diet enhanced host resistance to P. brasiliensis compared with the diet containing 20% casein. In this study, mice refed on 1.5% and 0% casein diets after 2d starvation showed normal serum total protein levels at least until day 7 after infection (Fig. 2B), indicating that these animals were not protein-malnourished during this period. Hu

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A

B

C

D

E

F

G

H

Fig. 4. Liver mRNA levels of myeloperoxidase (A), cathepsin-G (B), elastase-2 (C), interleukin-18 (D), chemokine C-X-C motif ligand 10 (E), nuclear factor-kB (F), inducible nitric oxide synthase (G), and granulocyte–macrophage colony-stimulating factor (H) in Paracoccidioides brasiliensis–infected mice refed diets with three levels (0%, 1.5%, or 20%) of casein. Transcript levels for each gene are expressed as relative mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. Each value represents mean 6 SD (n ¼ 5/test group at each day). Day 0, before infection/refeeding. Values highlighted with different superscripts (a–c) at the same days are significantly different (P < 0.05). Values highlighted with asterisks are significantly different from day 0 (P < 0.05).

et al. [7] reported that HBV expression is substantially inhibited and HBV-induced liver injury is greatly reduced in HBV transgenic mice fed a diet containing 6% casein for 3 mo compared with mice consuming a 22% casein diet. It

has been reported also that complete protection against malaria was achieved in mice fed a diet containing 0%, 5%, or 12.5% protein for 1 wk before infection compared with mice fed a 25% protein diet [8]. Li et al. [9] reported that

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Fig. 5. Spleen mRNA levels of myeloperoxidase (A), cathepsin-G (B), elastase-2 (C), and inducible nitric oxide synthase (D) in Paracoccidioides brasiliensisinfected mice refed diets with three levels (0%, 1.5%, or 20%) of casein. Transcript levels for each gene are expressed as relative mRNA levels (per glyceraldehyde-3-phosphate dehydrogenase mRNA). Each value represents mean 6 SD (n ¼ 5/test group at each day). Day 0, before infection/refeeding. Mean values highlighted with different superscripts (a,b) at the same days are significantly different (P < 0.05). Values highlighted with asterisks are significantly different from day 0 (P < 0.05).

mice fed on a 5% protein diet for 2 wk showed a prominent prolongation of survival compared with mice fed a diet containing 25% protein when both groups were injected with EL4 (T-cell lymphoma) and 3LL (lung carcinoma) tumor cells. A low-protein, low-caloric diet (9% of calories from protein) reduces human plasma levels of insulin-like growth factor-I, which promotes tumor development by stimulating cell proliferation and inhibiting cell death, when compared with a Western diet (16% of calories from protein) [18]. Taken together, it is conceivable that in certain cases protein restriction without malnutrition could enhance rather than suppress host resistance. Several studies have suggested that reduced productions of IFN-g and proinflammatory mediators are associated with reduction in host resistance to infection [19,20]. In our study, by contrast, spleen and liver levels of IFN-g and IL6 and liver mRNA levels of proinflammatory mediators increased less profoundly, whereas the fungal clearance in the spleen and liver were enhanced, after infection in mice refed on a diet containing 1.5% or 0% casein compared with those animals refed the 20% casein diet (Figs. 3A–D, 4A–H). Thus, there was no positive correlation between antifungal activity and production of any one of the proinflammatory mediators. We cannot offer any satisfactory explanation for these results except for speculating that the reduced level of IFN-g and

proinflammatory mediators in mice refed the 1.5% casein diet may be sufficient to resist the P. brasiliensis infection. Ling et al. [21] demonstrated that IL-6–mediated signaling pathways, Janus Kinase-Signal transducer and activator of transcription and mitogen-activated protein kinase pathways, are well preserved in livers of rats fed a 2% casein diet for 14 d, whereas serum albumin and total protein levels were significantly reduced in these animals. The 1.5% casein diet–induced decrease in IFN-g production might be due to decreased fungal loads as suggested by Rice et al. [22], who observed that serum IFN-g levels increased less profoundly, whereas bacterial clearance in the liver and spleen was enhanced, after Listeria monocytogenes infection in sympathectomized mice compared with non-treated controls. Ashman et al. [23] reported that Candida albicans infection increases IFN-g mRNA levels in mouse brain in proportion to the magnitude of the infectious burden, and the increased response of IFN-g then contributes to the clearance of yeasts from the brain. The mechanism(s) underlying the enhancing effects of low-protein diets on host resistance to P. brasiliensis and other micro-organisms remains to be elucidated. There is mounting evidence that overproduction of proinflammatory mediators causes detrimental effects. Overproduction of helper T-cell 1 cytokines such as IFN-g and IL-18 is responsible for the increased lethality observed in

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mice infected with C. albicans and Toxoplasma gondii [24,25], and treatment with an anti–IFN-g antibody improves the survival of mice infected with gram-negative bacteria [26]. IL-18 injection increased the mortality of burned mice with a mild Escherichia coli infection [27]. It has been proposed that IL-6, although critical to establishing antimicrobial defense, contributes to pathogenesis when released in excess [28]. In addition, excess production of cathepsin-G and elastase-2 in the lungs plays a central role in the pathology of inflammation pulmonary disease [29,30]. Overproduction of nitric oxide by iNOS is highly toxic to host cells [31]. In the present study, elevated levels of liver and spleen proinflammatory mediators were observed in infected mice refed the 20% casein diet compared with those refed the 1.5% or 0% casein diet. Taken together, refeeding with a conventional diet (such as that containing 20% casein) subsequent to acute starvation presumably might be more detrimental to host tissues compared with diets containing 1.5% or 0% casein and lead to a reduction in host resistance to infections. In the present study, we examined the effects of a shortterm (7 d), but not long-term, feeding of the 1.5% or 0% casein diet on host resistance. As for long-term feedings, Sakamoto et al. [32] reported that in rats fed the 0.5% casein diet for 8 wk, serum complement level was not significantly decreased but tuberculin skin reactivity was reduced compared with rats fed the 18% casein diet. Moriguchi et al. [33] reported that feeding the 5% casein diet for 7 wk had no significant effects on the abilities of alveolar macrophages to phagocytose latex and yeast cells, but mean body weight and numbers of alveolar macrophages of rats fed the 5% casein diet for 7 wk were about 65% and 27% of those fed the 20% casein diet, respectively. The long-term consequences of very low-protein diets (e.g., 1.5% casein diet) on host resistance to P. brasiliensis remain to be further investigated. Conclusion Our results suggest that protein restriction without malnutrition could enhance host resistance to P. brasiliensis, through reduced but sufficient production of proinflammatory mediators. References [1] Muramatsu K, Ashida K. Effect of dietary protein level on growth and liver enzyme activities of rats. J Nutr 1962;76:143–50. [2] Omran ML, Morley JE. Assessment of protein energy malnutrition in older persons, part II: laboratory evaluation. Nutrition 2000;16:131–40. [3] Nikawa T, Odahara K, Koizumi H, Kido Y, Teshima S, Rokutan K, et al. Vitamin A prevents the decline in immunoglobulin A and Th2 cytokine levels in small intestinal mucosa of protein-malnourished mice. J Nutr 1999;129:934–41. [4] Omran ML, Morley JE. Assessment of protein energy malnutrition in older persons, part I: history, examination, body composition, and screening tools. Nutrition 2000;16:50–63. [5] Cunningham-Rundles S, McNeeley DF, Moon A. Mechanisms of nutrient modulation of the immune response. J Allergy Clin Immunol 2005;115:1119–28.

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