key: cord-0731005-36b4twh6
authors: Bass, DM
title: Interferon gamma and interleukin 1, but not interferon alfa, inhibit rotavirus entry into human intestinal cell lines
date: 1997-07-31
journal: Gastroenterology
DOI: 10.1016/s0016-5085(97)70083-0
sha: 555f37e69df52aee3fddd1f56b388f8e45ddefb2
doc_id: 731005
cord_uid: 36b4twh6

Abstract BACKGROUND & AIMS: Rotavirus, an important agent of gastroenteritis in children, causes diarrhea by infecting differentiated villus enterocytes in the small intestine. The aim of this study was to determine whether cytokines that can be expressed by mucosal cells have an effect on the rotavirus susceptibility of cultured human enterocytes. METHODS: Caco-2 and HT-29 cells were pretreated with various cytokines before challenge with rotavirus. RESULTS: Interleukin (IL)-1, interferon (IFN)-alpha, and IFN-gamma pretreatment led to a dose-dependent resistance to rotavirus infection. Maximum effects occurred after 72 hours of pretreatment, whereas no detectable inhibition occurred with <12 hours of pretreatment. Liposomal transfection of single-shelled and double-shelled rotavirus particles bypassed the block to rotavirus replication in IFN-gamma- and IL-1- treated but not IFN-alpha-treated cells. Binding studies with purified, metabolically labeled rotavirus showed no significant difference among IFN-gamma- and IFN-alpha-treated and control Caco-2 cells. Viral entry into Caco-2 cells was significantly inhibited by IFN-gamma and IL-1 but not IFN-alpha. CONCLUSIONS: IFN-alpha and IFN-gamma induce rotavirus resistance by different mechanisms, suggesting that cytokines play a role in host defense against viral agents by changing the phenotype of intestinal epithelial cells. (Gastroenterology 1997 Jul;113(1):81-9)

is available regarding nonspecific host defense mechaof pretreatment, whereas no detectable inhibition ocnisms against rotavirus that may be important in precurred with õ12 hours of pretreatment. Liposomal transfection of single-shelled and double-shelled rotavirus parventing severe disease in naive hosts. A variety of cytoticles bypassed the block to rotavirus replication in kines, particularly the IFNs, have been shown to mediate IFN-g-and IL-1-treated but not IFN-a-treated cells.

Binding studies with purified, metabolically labeled rotavihost defense against mucosal viral pathogens has not been rus showed no significant difference among IFN-g-and well characterized.

In this study, the effects of various cytokines on the cluding the ability to form polarized monolayers with well-developed microvilli, expression of digestive en-T zymes such as sucrase-isomaltase and lactase, and the he intestinal epithelium serves as a barrier, protecting the host from noxious organisms and moleability to transport water and ions toward the basolatcules and simultaneously facilitating absorption of esseneral aspect. 8 They are also highly permissive for rotavitial nutrients. Recently a number of investigators have rus. 9 Whether cytokines that are known to be secreted shown that enterocytes are active participants in intercelby mucosal mononuclear or epithelial cells modulate lular cross-talk via cytokines with immune effector cells the susceptibility of enterocytes for rotavirus infection such as mononuclear cells and neutrophils. This interacwas examined. tion allows localized and specific modulation of epithelial

and immune effector responses under varying conditions. For example, enterocytes release various cytokines, in-Cytokines cluding interleukin (IL)-8, in response to invasive bacte-All cytokines were purchased as lyophilized powders ria, 1,2 presumably to recruit neutrophils to sites of bacteshipped, aliquoted, and stored according to the suppliers' recrial invasion. On the other hand, many cytokines originating in mononuclear cells, such as interferon Particles with methionine-free medium containing 100 mCi/mL [ 35 S]-Single-shelled virus preparations were treated with 10 methionine. HT-29 cells were a gift from John Barnard, Vanmmol/L ethylenediaminetetraacetic acid (EDTA) before use to derbilt University. Caco-2 cells were obtained from American ensure removal of outer capsid proteins. Such EDTA-treated Type Tissue Collection (Rockville, MD). Cells were grown in preparations had no residual infectivity when inoculated on RPMI medium supplemented with 10% fetal calf serum, L-MA 104 cells without liposomes. Monolayers were washed glutamine, penicillin, and streptomycin in a 5% CO 2 incubawith serum-free media and incubated at 37ЊC for 4 hours tor. Cells were grown in 96-or 24-well culture dishes. Unless with a mixture containing 10 mg/mL Lipofectin (GIBCO/BRL, otherwise stated, the cells were treated for the indicated times Gaithersburg, MD) and dilutions of CsCl-purified rotavirus with cytokines at the stated concentrations in fresh media after double-or single-shelled particles in serum-free media as dereaching confluence 4 days after plating. Control cells were scribed previously. 12 The cells were then fed an equal volume given fresh media without cytokine at the same time. Caco-2 of complete (10% fetal bovine serum) media and incubated cells, which were used 20 days after confluence for studies of overnight at 37ЊC. Infection was detected by immunoperoxidifferentiation effects, were fed fresh media every other day. dase staining.

All cytokine treatments were tested for cytotoxicity on

Caco 2-cells on 96-well plates by microscopic inspection for morphology, trypan blue exclusion, and MTT dye reduction. Confluent Caco-2 monolayers in 24-well dishes were Cells treated with maximal doses (125 U/mL) of IFN-g and washed twice and chilled to 4ЊC. Metabolically [ 35 S]-IFN-a for 72 hours were pulse-labeled with [ 35 S]methionine, methionine-labeled RRV (100,000 cpm/well, approximately and total counts incorporated were compared with those of 10 6 peroxidase focus units [pfu]/well, 10 pfu/cell) was added, medium-treated cells. Specific protein incorporation was also and the monolayers were incubated at 4ЊC with gentle rocking examined by lysing the cells with Laemmli's sample buffer for 1 hour. For measurement of binding, the monolayers were and performing sodium dodecyl sulfate-polyacrylamide gel washed three times with cold serum-free media, lysed with electrophoresis (SDS-PAGE) with fluorography.

2% SDS, and counted as previously described. 12 Under these conditions, approximately 10%-20% of the radiolabeled virus

bound to the monolayer. Internalization was determined by binding the 35 S-labeled virus as described, followed by warm-Monolayers in 96-well plates were washed with serumfree media and inoculated with dilutions (1:5000) of trypsin-ing to 37ЊC for 120 minutes. The cells were then treated with trypsin-EDTA (101 solution; Sigma) in saline for 30 minutes activated virus at a concentration calculated to equal approximately 100 infectious units per well. Rotavirus stock was acti-at 4ЊC, washed twice with ice-cold media containing 2 mmol/ L phenylmethylsulfonyl fluoride and 10% fetal calf serum, vated by treating with trypsin (type VIII; Sigma) at 5 mg/mL for 30 minutes at 37ЊC. Infection was allowed to proceed lysed, and counted. Control experiments showed that 95% of virus bound at 4ЊC was removed under these conditions if the overnight at 37ЊC. Infected cells were identified by immunoperoxidase staining as previously described. 11 Briefly, mono-monolayers were not warmed.

/ 5e1e$$0040 06-10-97 13:01:28 gasa WBS-Gastro responses in cultured intestinal epithelial cells 3,4,13 before challenge with rotavirus. IL-8 was also tested because it is released by intestinal epithelia after infec-ment. None of the cytokines tested showed any evidence of toxicity to the cells at the concentrations used tion with rotaviruses 14 and invasive bacteria. 2 The relatively long pretreatment period, 72 hours, was cho-by gross morphology, trypan blue exclusion, or MTT reduction. Neither IL-1 nor IFNs had an effect on total sen to provide maximum sensitivity in detecting cytokine effects on Caco-2 susceptibility to rotavirus. The counts per minute (cpm) of [ 35 S]methionine incorporated into the Caco-2 cells. In addition, treatment with results, summarized in Table 1 , indicate that IL-1a and IL-1b induced a moderately rotavirus-resistant IFN did not cause any gross change in the specific SDS-PAGE pattern of metabolically labeled Caco-2 state in the Caco-2 cells in a dose-dependent manner (maximum inhibition, 70% of control), whereas IL-6 proteins (data not shown). Additional studies were performed to characterize led to a reproducible decrease in infection of dubious physiological significance (maximum inhibition, the time course and nature of the antiviral effect. Figure 1 shows the effect of different durations of IFN-a 30%). Although synergy of effects of IL-1 and IL-6 in the expression of acute-phase proteins by Caco-2 cells and IFN-g pretreatment on rotavirus resistance in a typical experiment. Six-hour IFN treatment of Caco-has been observed, 13 no additive effects were noted when the cells were treated with both cytokines in 2 cells had no significant effect on rotavirus infection. By 24 hours, rotavirus resistance was evident but re-varying concentrations in a ''checkerboard'' simultaneous titration (data not shown). IFN-g and IFN-a pre-quired higher concentrations of IFN-g (mean inhibitory concentration [MIC 50 ], 40 U/mL) compared with treatment led to significant dose-dependent resistance to rotavirus infection (maximum inhibition, 99% and an MIC 50 of approximately 3 U/mL for 72 hours of treatment. The resistance to rotavirus largely persisted 90%, respectively). Similar results were obtained when cytokine-pretreated HT-29 cells were challenged with even if cytokine was removed for 48 hours from cells that had been previously treated for 72 hours. Similar rotavirus (Table 1) . IFN-and IL-1 -induced rotavirus resistance was completely abrogated by the addition time course results were obtained for IL-1 treatment of Caco-2 cells (data not shown). Because rotavirus in of appropriate neutralizing antibodies but not affected by addition of a control antibody (data not shown).

vivo typically infects the most differentiated villus tip cells of the small intestine and because IFNs can affect IFN-and IL-1 -induced rotavirus resistance was also abrogated by the presence of cycloheximide (5 mg/mL) cell differentiation, we studied the effect of the state of differentiation of the Caco-2 cells on the antiviral during the first 18 of 24 hours of IFN or IL-1 treat-/ 5e1e$$0040 06-10-97 13:01:28 gasa WBS-Gastro state induced by IFN-g and IFN-a. The differentiation were thus transfected into the IFN-g-treated Caco-2 cells, there was virtually no inhibition of viral replication of Caco-2 cells toward a phenotype characteristically increases similar to differentiated villus tip enterocytes within the range of IFN-g concentrations tested ( Figure  2A ). Because production of new viral antigen was being with time after plating. 15 In our study, differentiated (20-day) Caco-2 cells expressed approximately 10-fold measured with the immunoperoxidase staining assay, the lack of inhibition observed indicates that under these more alkaline phosphatase than the undifferentiated (4-day) Caco-2 cells. We treated monolayers of Caco-conditions, IFN-g treatment had no significant effect on rotavirus transcription or translation in Caco-2 cells. 2 cells either 4 days (early confluence) or 20 days (differentiated) after plating with various concentrations Double-shelled, infectious rotavirus virions become transcriptionally activated when they lose their outer cap-of IFN and IL-1 for 48 hours before challenge with Wa rotavirus. Both IFNs and IL-1 induced rotavirus sid. 17 Uncoating is therefore an essential early step in the rotavirus replicative cycle. If an uncoating defect was resistance in Caco-2 cells regardless of their differentiation status. The more differentiated cells were consis-present in the IFN-treated Caco-2 cells, it would be expected that double-shelled particles are unable to infect tently significantly more sensitive to IFN-g at lower concentrations of IFN tested (2 -4 U/mL). No differ-the cells. To determine whether viral uncoating was inhibited by IFN-g, similar liposomal transfections were ences were noted in the effects of IFN-a or IL-1 on the two populations of Caco-2 cells. Interestingly, performed with double-shelled rotavirus particles ( Figure  2B ). Like single-shelled particles, double-shelled parti-there were no significant differences in the rotavirus susceptibility of the media-treated control 4-day and cles were fully infectious in IFN-g-treated cells if they were introduced into the cells via liposomes.

Caco-2 cells (Figure 3 ). Successful rotavirus lipofection Caco-2 Cells of single-and double-shelled rotavirus particles was Because most studies on the IFN-induced antivialso performed in IFN-g -and IL-1 -treated HT-29 ral state have described inhibition of viral transcription cells (data not shown). or translation, 16 

Lipofection of Single-Shelled Rotavirus also limited rotavirus replication at either of these steps Particles Fails to Bypass Rotavirus of the viral life cycle was determined. Cationic liposomes

Resistance of IFN-a-Treated Caco-2 Cells were used to transfect viral particles into cells, a method previously used to induce productive rotavirus infection Because IFN-a was also a potent inhibitor of rotavirus replication (Table 1) , single-shelled rotavirus in naturally nonpermissive murine L-cell fibroblasts. 12 When single-shelled, noninfectious rotavirus particles particles were transfected into IFN-a -treated cells in / 5e1e$$0040 06-10-97 13:01:28 gasa WBS-Gastro an identical fashion. Unlike IFN-g -pretreated cells, failed to bypass IFN-a rotavirus inhibition in Caco-2 or HT-29 cells (data not shown).

Neither IFN-g, IL-1, nor IFN-a Has a (Figure 4 ). Double-shelled rotavirus particles also Significant Effect on Rotavirus Binding to Caco-2 Cells

Because uncoating, transcription, and translation were not blocked in the IFN-g-and IL-1-treated cells, it was deduced that inhibition must be caused by a very early step in viral replication. To identify this early step, binding studies were performed using purified, metabolically labeled RRV rotavirus. Results of a typical experiment are shown in Table 2 . No significant difference in radiolabeled virus binding between control and IL-1-, IFN-a-, and IFN-g-treated cells could be observed. Because many of the purified rotavirus particles may not be capable of initiating infection, it was possible that IFN reduced the binding of the infectious fraction of rotavirus particles. Therefore, the binding of infectious particles of both RRV and Wa rotavirus was also measured using previously described methods. 12 As can be seen from Table 2 , this approach failed to show any 

Results are expressed as percentage of media-treated control wells that were infected in the same manner (double-shelled particles or

Lipofectin/single-shelled particles).

kines, particularly IL-1, IFN-a, and IFN-g, could downregulate the rotavirus permissivity of Caco-2 cells ( Table  1) . The experiments cannot exclude the possibility that virus was allowed to bind to IL-1-, IFN-g-, IFN-a-, other cytokine-like substances could be released by enand mock-treated monolayers. After 1 hour of binding terocytes in response to IFN or IL-1 and mediate the at 4ЊC, the monolayers were washed and warmed to 37ЊC for 2 hours. Surface-bound virus was removed with trypantiviral effects. sin-EDTA, and internalized counts per minute were de-

The time course of development of the antiviral state termined by scintillation counting. The results showed (onset at approximately 12 hours, peak at 72 hours; Fig-90% inhibition of rotavirus internalization in cells that ure 1) was similar to that observed with the development had been pretreated with 75 U/mL IFN-g ( Figure 1 ).

of IFN-g-or IL-4-induced changes in enterocyte pheno-This is quite similar to the level of inhibition of viral type such as decreased stimulated Cl 0 secretion and mareplication found with similar doses of IFN-g ( Figure 2 ) jor histocompatibility complex expression. 3, 4 This time and suggests that poor internalization of virus is the course is also compatible with previously described IFNmajor block to rotavirus replication in IFN-g-treated induced antiviral states. It is not surprising to observe that IFNs induce strong antiviral states in Caco-2 cells, given that IFN was originally discovered, described, and named for its antiviral effect. 18 Type I IFNs (such as IFN-a) are believed to inhibit viral translation by several pathways including inactivation of the eukaryotic initiation factor 2a by phosphorylation via an induced kinase. Thus, in early studies of type I IFN-induced antiviral states, transfection of viral RNA failed to cause cellular infection. 19 Previous experiments in which liposomal transfection of rotavirus failed in IFN-a-treated Caco-2 cells confirm those results (Figure 4) .

The antiviral properties of IFN-g (type II IFN) have been studied less than those of the type I IFNs such as IFN-a. IFN-g binds to a distinct receptor and apparently induces a distinct antiviral state. [19] [20] [21] [22] [23] The fact that anti- (Figures 2 and 3) . Because this procedure redissolved with Laemmli's sample buffer, and residual radioactivity was determined by scintillation counting.

sulted in control levels of transcription and translation of new viral antigen as detected by our immunoperoxidase assay, it can be concluded that inhibition of viral transcription and/or translation is not important in the toskeletal elements, 31-33 either of which may be relevant IFN-g-or IL-1-induced rotavirus resistance. The proto this mechanism of cellular resistance. We have preduction of new viral antigen by transfected cells also viously described modification of cellular susceptibility shows that the antiviral effect was not caused by nonspeto rotavirus by cytoskeletal inhibitors. 28 In these studies, cific cytotoxic effects of the cytokines. Studies of viral cytochalasin B and D were capable of enhancing susceptibinding and entry into cytokine-treated cells showed that bility of normally resistant murine L cells to rotavirus. most of the IFN-g-and IL-1-induced viral resistance Neither cytochalasins, Taxol, nor cholcine had any ability can be accounted for by failure of bound rotavirus to to inhibit rotavirus infection of permissive cell lines. enter the cell ( Figure 5 ).

Our studies do not directly address the in vivo role of IFN-induced resistance to entry of invasive bacteria IFN and other cytokines in host defense against enteric has been noted previously. 25 Whitiker-Dowling et al. viral pathogens. In previous human and animal studies described type I IFN-induced reduction of vesicular stoof rotavirus gastroenteritis, type I IFN was detected both matitis virus cell entry in some (but not all) murine locally and systemically at levels comparable to or higher fibroblast cell lines, but the effect was much less prothan those used in these experiments. [34] [35] [36] Exogenous nounced and required much higher doses of IFN. 26, 27 IFN-a has been reported to be therapeutic in rotavirus Thus, a potent and novel mechanism for an IFN-g-indiarrhea in piglets and calves. 37,38 IL-1 has been found duced antiviral state may be applicable to other cells and in both normal and inflamed intestinal tissue at levels viruses in the present study. In fact, IFN-g-pretreated ranging from 0.7 ng/mL for normal tissue to 50 ng/mL Caco 2 cells are equally resistant to astrovirus, a comin tissue from active colitis. 39 Elevated intestinal levels pletely unrelated agent of viral diarrhea, and serotype 1 of IL-1 messenger RNA have been detected during rota-(Lang) reovirus (D. Bass, unpublished observations).

virus infection of mice in a number of murine strains (R. The barrier to cell entry of rotavirus in IFN-g-and Shaw, personal communication, December 1996). Basal IL-1-treated Caco 2 cells is unclear. Rotaviruses (unlike levels of IFN-g similar to those used in this study are astroviruses and reoviruses) infect cells by an endocytosisreadily detectable in noninflamed intestinal tissues. 40 Viindependent direct membrane penetration. 11,12,28 IFN-g ral infection could induce increased intestinal levels of has been reported to induce changes in cellular mem-IFN-g in vivo by natural killer cells or rotavirus-specific T cells that have been shown to secrete IFN-g in the brane lipid composition 29,30 and changes in cellular cy-

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Intestinal immune responses in hu-The author thanks Usha Upadhyayulla for technical assistance