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,2,**
,3
Division of Nutritional Sciences, Departments of
*
Animal Sciences,
**
Food Science and Human Nutrition, and
Veterinary Pathobiology, University of Illinois at Urbana-Champaign and
Mead Johnson Research Center, Evansville, IN
3To whom correspondence should be addressed.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rotavirus • inflammation • malnutrition • pigs • small intestine
|
INTRODUCTION |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kapikian and Chanock 1996
). Extensive child morbidity and mortality continue
nonetheless. In developing countries, in which nutritional status is
often compromised, rotaviruses cause >850,000 deaths each year.
Worldwide, rotaviruses account for >25% of diarrheal-related deaths
and 6% of all deaths in children <5 y old (Matsui and Angel 1997
). Considerable promise arises from recent efforts to
develop an antirotavirus vaccine (Glass et al. 1996
). In
fact, progress with vaccine strategies exceeds our understanding of
mechanisms underlying rotavirus pathophysiology. Knowledge of the
molecular and cellular basis of host responsiveness to rotavirus
infection remains crucial, particularly in the context of malnutrition,
which exacerbates disease severity and will likely affect the efficacy
of antirotavirus vaccine strategies.
Rotavirus infections are characterized by viral replication in small
intestinal enterocytes (Estes 1990
), with subsequent
cell lysis and attendant villous blunting (Theil et al. 1978
), depressed levels of mucosal disaccharidases
(Bishop et al. 1973
), watery diarrhea (Theil et al. 1978
) and dehydration. It is generally accepted that the
destruction of villous epithelial cells reduces enzymatic and
absorptive capacity in the small intestine, resulting in a
malabsorptive-type diarrhea (Graham et al. 1984
). With a
neonatal pig model, we recently identified nutritionally responsive
indices of structural repair that remain operative despite epithelial
damage in rotavirus-infected intestine (Zijlstra et al. 1997
). Those findings bring into question the extent to which
malabsorption contributes to rotaviral diarrhea and challenge the
common nutritional management protocol of "bowel rest," i.e.,
reduction of luminal nutrients during the diarrheal episode
(American Academy of Pediatrics 1996
, Lieberman 1994
, Zijlstra et al. 1997
). We have now
extended our observations and provide evidence that rotavirus induces
an attendant intestinal inflammatory response that may contribute to a
secretory-type diarrhea. Also identified are molecular and biochemical
indices of rotavirus infection that are responsive to malnutrition,
enabling opportunities to understand how individual macronutrients
contribute at the intestinal level to host clearance of rotavirus.
|
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal protocol was approved by the University of Illinois Laboratory
Animal Care Advisory Committee and followed principles established by
the NIH (NRC 1985
). Experimental design, diet
considerations, and infection protocol were described in more detail in
a previous report of other data from this study (Zijlstra et al. 1997
). Two-d-old cesarean-delivered, colostrum-deprived piglets
(n = 39) were randomly assigned to three treatment
groups as follows: 1) noninfected, fully nourished;
2) infected, fully nourished; and 3)
infected, malnourished. Pigs in groups 2 and 3 were infected with
porcine rotavirus at 2 d of age.
All piglets were fed a liquid diet formulated to meet the nutritional
requirements for growing piglets from birth through 3 wk of age
(McClead et al. 1990
) and prepared by the Mead Johnson
Nutritional Group (Evansville, IN) as a dry powder. Pigs in groups 1
and 2 were fed complete reconstituted formula (180 g/L) and were
pair-fed according to daily intake of the infected, fully nourished
group. For the malnourished, infected group, formula was diluted 50%
with water plus electrolytes and also supplied according to daily
volume intake of infected, fully nourished piglets (group 2)
(Butzner et al. 1985
, Zijlstra et al. 1997
).
Diarrhea was scored daily based on consistency of feces (0, no diarrhea; 1, stiff flowing feces; 2, easy flowing feces; 3, severe, watery diarrhea). On d 2, 9 and 16 postinfection, four piglets per treatment were killed by electrocution followed by exsanguination. The small intestine was dissected free of mesentery and arranged in six parts of equal length to enable collection of tissue at seven equidistant sites from the duodenum (segment 1) and proximal jejunum (segment 2) to distal jejunum (segment 6) and distal ileum (segment 7). Small intestine tissue was collected immediately and fixed for immunocytochemistry or frozen at -80°C for other analyses as described below.
Intestinal MHC class I and II expression.
Intestinal major histocompatibility complex
(MHC)4
RNA expression was monitored as a general barometer of inflammation
(Abbas et al. 1991
). Total RNA was extracted from frozen
jejunal tissue (segment 6) using the acid guanidinium
thiocyanate-phenol-chloroform method (Chomczynski and Sacchi 1987
). Total RNA samples (10 µg each) were size-separated in
1.25% agarose/3% formaldehyde gels and immobilized onto nylon
membranes (Magna Graph, Westborough, MA) by using standard Northern
blotting techniques (Sambrook et al. 1989
). Blots were
probed with 
32P-labeled cDNA probes specific for porcine
MHC class I and II genes (kindly provided by Dr. L. B. Schook,
University of Minnesota) with the use of conventional hybridization
technique (Sambrook et al. 1989
). To verify equivalent
RNA loading in agarose gels, blots were probed with an
32P-labeled cDNA specific for human ß-actin.
Autoradiographic exposure of the membranes to Kodak (Rochester, NY)
X-Omat AR film was carried out at -80°C with intensifying screens.
Relative signal intensities were determined by laser densitometry using
ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Lymphocyte immunocytochemistry.
Intestinal CD4+ and CD8+ T lymphocytes, which
participate in effector mechanisms underlying rotavirus immunity
(Bruce et al. 1995
, Matsui and Angel 1997
), were monitored in this study by quantitative
immunocytochemistry. Jejunal tissues (segment 5) were fixed in Bouin's
solution (Carson 1990
) for 24 h, soaked in four
changes of 70% ethanol to remove excess picric acid, then embedded in
paraffin and sectioned onto glass slides. Slides were deparaffinized in
xylene and then rehydrated through a series of washes in alcohol, water
and Tris buffer. Fluorescent anti-pig CD4 (clone 74–12-4) or
biotinylated anti-pig CD8 (clone 76–2-11) antibodies were used
according to standard immunofluorescence or immunocytochemical
techniques (Carson 1990
, Saalmuller 1996
). Tissue sections were blocked with 20% mouse serum
before primary antibody incubations for 2 h at 37°C.
Biotinylated anti-CD8 positive cells were developed using a Histomark
Red kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD) according
to the manufacturer's protocol. Electronic images were captured using
a Sony (New York, NY) PowerHAD color video camera attached to a
Nikon (Melville, NY) OPTIPHOT-2 microscope. CD4+ or
CD8+ lymphocytes were counted in one entire jejunal section
per pig at a magnification of X 100 (Olympus BH-2-RFCA, Lake Success,
NY). To standardize lymphocyte counts, entire jejunal cross-sectional
areas were measured using Image-Pro Plus software (Media Cybernetics,
Silver Spring, MD).
Intestinal prostaglandin E2 (PGE2) analysis.
Approximately 1 g of frozen jejunal tissue (segment 4) was
homogenized on ice in 2 mL of 95% methanol for 30 s. Homogenates
were centrifuged at 3000 x g for 15 min at 4°C
for supernatant recovery. PGE2 was measured with an enzyme
immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI) according to the
manufacturer's protocol. EIA plates were read on a
Thermomax microplate reader (Molecular Devices, Menlo Park,
CA). To normalize jejunal PGE2, total protein
concentrations in the methanol-insoluble fraction were determined by
the modified Lowry method (Hartree 1972
) with bovine
serum albumin as a standard.
Statistical analyses.
Data were analyzed using the General Linear Models procedure of the SAS
statistical package (SAS 1985
). Infected, fully
nourished piglets (group 2: d 2, n = 5; d 9,
n = 5; d 16, n = 4) were
compared by preplanned contrasts with either noninfected piglets (group
1: d 2, n = 4; d 9, n = 4; d
16, n = 4) to determine the effect of rotavirus
infection, or to infected, malnourished piglets (group 3: d 2,
n = 4; d 9, n = 5; d 16,
n = 4) to determine the effect of malnutrition
within infected piglets (Steel and Torrie 1980
). Results
are presented as least-square means ± pooled
SEM. Differences were considered significant when
P < 0.05. Instances in which P < 0.1 are discussed as trends. To consider possible mechanisms
underlying intestinal responses to rotavirus alone, or to the combined
effects of rotavirus and malnutrition, relationships among diarrhea
observations and inflammatory variables were evaluated by Spearman
correlation analysis (SAS 1985
).
|
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nutrient intake and weight gain data were presented in a previous
report of this study that focused on animal growth and metabolic and
small intestinal parameters of recovery from rotavirus (Zijlstra et al. 1997
).
Diarrhea was not observed in any treatment group before inoculation
with rotavirus (Fig. 1
). Rotavirus infection resulted in severe, watery diarrhea within
24 h, which lasted for 1 wk for all infected piglets, regardless
of nutritional regimen (Fig. 1)
. By 8 d postinfection, diarrhea
began to subside in both nourished and malnourished piglets. Diarrhea
cleared completely by 10 d postinfection in infected, nourished
piglets (Fig. 1)
. In contrast, diarrhea continued through d 16
postinfection for infected, malnourished pigs, although the physical
appearance of excreted material changed from a liquid consistency to a
more paste-like, but easy flowing consistency (Fig. 1)
.
|
). Nourished, infected piglets began to regain body weight
by 5 d postinfection, with rate of body weight gain similar to
that of noninfected piglets at 11 d postinfection (Zijlstra et al. 1997
). In contrast, after the initial 48-h period of
weight loss, body weights of infected, malnourished piglets remained
essentially static throughout the postinfection period. Intestinal MHC class I and II RNA expression.
At 2 d postinfection, the level of MHC class I RNA expression
doubled in the distal jejunum of rotavirus-infected, nourished piglets
relative to noninfected piglets (Fig. 2
; P < 0.01). A similar increase in intestinal MHC
class I RNA expression was observed for rotavirus-infected,
malnourished piglets (Fig. 2
; P < 0.1 vs. infected,
nourished). At 9 d postinfection, MHC class I RNA expression
increased further for both nutritional regimens. The level of MHC class
I RNA expression in the jejunum of infected, nourished piglets was
three times that observed for noninfected pigs (Fig. 2
;
P < 0.001). Among rotavirus-infected piglets on d 9,
malnutrition resulted in an additional 26% increase in intestinal MHC
class I RNA expression (Fig. 2
; P < 0.01). At 16 d postinfection, jejunal MHC class I RNA expression was not different
(P > 0.1) among treatment groups; however the level of
expression was numerically greater in infected, malnourished than in
infected, nourished piglets (Fig. 2)
.
|
. Rotavirus infection doubled distal jejunal MHC class II RNA
expression 2 d postinfection in nourished piglets
(P < 0.05). In contrast, malnutrition prevented the
apparent normal increase in intestinal MHC class II expression in
response to rotavirus 2 d postinfection. At 9 d
postinfection, a comparable level of constitutive MHC class II RNA
expression was observed for the three treatment groups
(P > 0.10). Similarly, significant differences were
not observed for intestinal MHC class II among treatments on d 16
postinfection; however, constitutive MHC class II expression was
numerically lower for infected, malnourished than for infected,
nourished piglets (Fig. 2)
. To gain additional perspective on the activation of MHC class I vs. class II gene expression in response to both rotavirus and superimposed malnutrition, the ratios of intestinal expression of MHC class I to class II were calculated. In response to rotavirus, the MHC class I/II expression ratio increased on d 2 and 9 postinfection for both nutritional regimens. The intestinal MHC class I/II ratios did not differ between noninfected and infected, nourished animals on d 16 postinfection. Because the level of MHC class I expression generally increased in response to malnutrition and the level of MHC class II was generally suppressed by malnutrition, the MHC class I/II ratio was numerically greater for infected, malnourished piglets relative to infected, nourished piglets throughout the study.
Intestinal CD4+ and CD8+ T lymphocytes.
The number of jejunal CD4+ T lymphocytes in
infected, nourished piglets was more than twice that of noninfected
piglets on d 2, three times greater on d 9 (P < 0.05),
and approximately doubled on d 16 (Fig. 3
). Generally, malnutrition did not affect the temporal pattern of
rotavirus-induced CD4+ T-lymphocyte expansion
(Fig. 3)
.
|
). A
numerical (P > 0.1) increase in jejunal
CD8+ T-lymphocyte numbers was also observed 9 and
16 d postinfection (Fig. 3)
. Similar to observations for
CD4+ T lymphocytes, malnutrition generally did
not affect the number of CD8+ T cells in the
jejunum of rotavirus-infected piglets (Fig. 3)
. The ratio of
CD4+ to CD8+ T lymphocytes
was similar on each of the days examined postinfection. Intestinal PGE2.
Rotavirus infection increased PGE2 concentrations
10-fold in the mid-jejunum of nourished piglets by 2 d
postinfection (P < 0.05; Fig. 4
). A sixfold increase in jejunal PGE2
concentrations was observed for infected, malnourished piglets (Fig. 4)
. At 9 d postinfection, PGE2
concentrations in infected, nourished piglets were not different from
noninfected animals and thus had returned to baseline. In contrast,
intestinal PGE2 concentrations in infected,
malnourished piglets remained elevated relative to infected, nourished
piglets (P < 0.05; Fig. 4
). Intestinal
PGE2 concentrations tended to be elevated in
infected, nourished piglets relative to noninfected piglets again on d
16 postinfection (P < 0.10; Fig. 4
). Intestinal
PGE2 concentrations remained elevated for
infected, malnourished piglets on d 16, with concentrations similar to
those of the infected, nourished group (Fig. 4)
.
|
Diarrhea observations were positively related to
CD4+ T-lymphocyte numbers (r =
0.31; P < 0.10), CD8+
T-lymphocyte numbers (r = 0.46; P < 0.01), MHC class I expression (r = 0.53;
P < 0.01) and PGE2
concentrations (r = 0.53; P <
0.001) (Table 1
).The number of jejunal CD4+ T lymphocytes
was positively correlated with CD8+ T-lymphocyte
numbers (r = 0.42; P < 0.01), MHC
class I expression (r = 0.32; P <
0.10) and PGE2 concentrations (r = 0.33; P < 0.05). The number of jejunal
CD8+ T lymphocytes was positively correlated with
MHC class I expression (r = 0.58; P <
0.001) and PGE2 concentrations (r = 0.39; P < 0.05). Jejunal MHC class I expression
was positively correlated with MHC class II expression
(r = 0.33; P < 0.10) and
PGE2 concentrations (r = 0.48;
P < 0.01).
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
This study suggests that the time-honored treatment method of "bowel
rest" after rotavirus infection may delay mucosal recovery.
Rotavirus infection is characterized by viral replication in small
intestinal enterocytes with subsequent cell lysis and attendant villous
blunting, depressed levels of mucosal disaccharidases, watery diarrhea
and dehydration (Bishop et al. 1973
, Estes 1990
, Theil et al. 1978
). Because of the reduced
enzymatic and absorptive capacity in the small intestine, it is
generally accepted that rotavirus elicits a malabsorptive-type of
diarrhea (Graham 1984
, Rhoads et al. 1991
). On the basis of clear evidence of local T-lymphocyte
expansion, enhanced intestinal MHC class I and class II gene expression
and elevated tissue concentrations of PGE2, we
propose that intestinal inflammatory responses to rotavirus may
contribute to a secretory-type of diarrhea.
In a previous report on this piglet model, we demonstrated that the
onset of rotaviral diarrhea coincided with villous destruction and
reduction of mucosal disaccharidase activities (Zijlstra et al. 1997
). Those observations agree with reports on other
experimental models of rotavirus infection and are consistent with the
possibility that malabsorption contributes to rotaviral diarrhea
(Zijlstra et al. 1997
). However, a clear distinction
between intestinal damage and diarrhea was established from the
observation that diarrhea subsided by 9 d postinfection in
nourished but not in malnourished piglets, whereas the degree of
intestinal structure damage was comparable between these nutritional
groups at that time postinfection (Zijlstra et al. 1997
). Thus, it is unlikely that malabsorption resulting from
epithelial damage is the sole explanation for rotaviral diarrhea.
Results from other model systems and limited observations from human
studies also invoke reconsideration of the pathophysiology of
rotavirus-induced diarrhea. At least three other reports on a piglet
model demonstrate rotavirus-induced diarrhea before extensive damage to
the intestinal epithelium (McAdaragh et al. 1980
,
Theil et al. 1978
). For example, piglets inoculated with
porcine rotavirus developed watery diarrhea 8 h after infection,
whereas minor damage to jejunal segments was not observed until 48 h postinfection (Vellenga et al. 1992
). Similarly, for a
mouse model, there was no correlation between the number of rotavirus
infected cells and the severity of diarrhea (Bass and Greenberg 1995
). Studies with a heterologous mouse model demonstrate that
chemically inactivated rhesus monkey rotavirus induces diarrhea in the
absence of epithelial attachment, cellular entry or viral replication
(Shaw at al. 1995
). It has also been reported that
diarrhea preceded obvious cell damage in human infants infected with
rotavirus (Bass and Greenberg 1995
). Among intestinal
biopsy specimens from 40 rotavirus-infected infants, only 5% exhibited
histologic evidence of damage (Kohler et al. 1990
).
New concepts of rotaviral pathogenesis are evoked most convincingly
from the recent identification of a rotaviral enterotoxin (Ball et al. 1996
, Dong et al. 1997
, Glass et al. 1996
). While making an antiserum to the nonstructural
rotaviral glycoprotein, NSP4, Estes and co-workers fortuitously
discovered that intraperitoneal delivery of purified NSP4 induced
diarrhea in a mouse model (Ball et al. 1996
). Subsequent
studies from this group with several model systems clearly demonstrate
that NSP4 alters Ca2+ homeostasis in host cells
through receptor-mediated phospholipase C activation and inositol
1,4,5-triphosphate production (Dong et al. 1997
). That
finding is consistent with numerous other examples of the mediation of
infectious diarrhea through altered intracellular
Ca2+ homeostasis, leading to
Ca2+-dependent fluid secretion across the mucosa
toward the lumen (Argenzio 1996
). Local prostaglandins
can also serve as mediators of intestinal ion imbalances, resulting in
epithelial chloride secretion (Kandil et al. 1994
). The
possible contributions of PGE2 to rotaviral and
specifically NSP4-induced diarrhea should be defined, considering the
present evidence that malnutrition exacerbates
PGE2 responses to rotavirus, together with clear
evidence that tissue PGE2 concentrations can be
altered through manipulation of diet fatty acid composition
(Fritsche et al. 1993
).
Prolonged diarrhea in infected, malnourished piglets was associated
with more intense and prolonged expression of local mediators or
markers of intestinal inflammation. Relative to infected, nourished
piglets, intestinal PGE2 concentrations were
greater and remained elevated longer in malnourished piglets also
infected with rotavirus. Similarly, patterns of intestinal MHC class I
gene expression, an acutely sensitive barometer of local inflammation,
were positively and significantly correlated with the pattern of
PGE2 expression and with prolonged diarrhea.
Those results are consistent with substantial evidence that
undernutrition typically intensifies viral enteritis and results in
greater morbidity and mortality from infections
(Cunningham-Rundles 1994
). The identification of
specific indices of rotavirus infection that are responsive to
malnutrition enables further studies to determine how individual
macronutrients contribute at the intestinal level to host clearance of
rotavirus. That information may facilitate the design of rational
nutritional therapies to enhance recovery from rotavirus infection, or
to maximize responses to rotavirus immunization protocols in geographic
regions subject to malnutrition.
|
FOOTNOTES |
|---|
2 Current address: North Carolina State
University, Box 7621, Raleigh, NC 27695-7621.
3 Abbreviations used: EIA, enzyme immunoassay;
MHC, major histocompatibility complex; PGE2, prostaglandin
E2.
Manuscript received September 7, 1998. Initial review completed November 23, 1998. Revision accepted January 7, 1999.
|
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J. W. A. Rossen, J. Bouma, R. H. C. Raatgeep, H. A. Buller, and A. W. C. Einerhand Inhibition of Cyclooxygenase Activity Reduces Rotavirus Infection at a Postbinding Step J. Virol., September 15, 2004; 78(18): 9721 - 9730. [Abstract] [Full Text] [PDF]
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N. J. Correa-Matos, S. M. Donovan, R. E. Isaacson, H. R. Gaskins, B. A. White, and K. A. Tappenden Fermentable Fiber Reduces Recovery Time and Improves Intestinal Function in Piglets Following Salmonella typhimurium Infection J. Nutr., June 1, 2003; 133(6): 1845 - 1852. [Abstract] [Full Text] [PDF]
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 G. M. Anstead, B. Chandrasekar, W. Zhao, J. Yang, L. E. Perez, and P. C. Melby Malnutrition Alters the Innate Immune Response and Increases Early Visceralization following Leishmania donovani Infection Infect. Immun., August 1, 2001; 69(8): 4709 - 4718. [Abstract] [Full Text] [PDF]
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R. Jiang, X. Chang, B. Stoll, M. Z. Fan, J. Arthington, E. Weaver, J. Campbell, and D. G. Burrin Dietary Plasma Protein Reduces Small Intestinal Growth and Lamina Propria Cell Density in Early Weaned Pigs J. Nutr., January 1, 2000; 130(1): 21 - 26. [Abstract] [Full Text]
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