Histone deacetylase inhibitor suppresses virus-induced proinflammatory responses and type 1 diabetes
Naoko Hara • Aimon K. Alkanani • Charles A. Dinarello • Danny Zipris
Abstract
Microbial infections are hypothesized to play a key role in the mechanism leading to type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced islet destruction to better understand how virus infection triggers T1D. Inoculation of the LEW1.WR1 rat with KRV results in systemic inflammation followed by insulitis and islet destruction 2–4 weeks post-infection. In this study, we evaluated the effect of treatment with the anti-inflammatory histone deacetylase inhibitor (HDACi) ITF-2357 on KRV-induced immunity and disease progression. Administering LEW1.WR1 rats with KRV plus ITF-2357 on 14 consecutive days beginning on the day of infection protected animals from islet infiltration and T1D. ITF- 2357 reversed KRV-induced T and B cell accumulation in the spleen or pancreatic lymph nodes on day 5 following infec- tion. Moreover, ITF-2357 reduced the expression level of KRV-induced p40 subunit of IL-12/IL-23 in spleen cells in vitro and in the peripheral blood in vivo. ITF-2357 suppressed the KRV-induced expression of transcripts for IRF-7 in the rat INS-1 beta cell line. ITF-2357 increased the virus-induced IL-6 gene expression in the spleen, but did not alter the ability of LEW1.WR1 rats to develop normal KRV- specific humoral and cellular immune responses and clear the virus from the pancreatic lymph nodes, spleen, and serum. Finally, ITF-2357 reversed virus-induced modulation of bac- terial communities in the intestine early following infection. The data suggest that targeting innate immune pathways with inhibitors of HDAC might represent an efficient therapeutic strategy for preventing T1D.
Key message
• Microbial infections have been implicated in triggering type 1 diabetes in humans and animal models.
• The LEW1.WR1 rat develops inflammation and T1D following infection with Kilham rat virus.
• The histone deacetylase inhibitor ITF-2357 suppresses virus-induced inflammation and prevents diabetes.
• ITF-2357 prevents T1D without altering virus-specific adaptive immunity or virus clearance.
• ITF-2357 therapy may be an efficient approach to prevent T1D in genetically susceptible individuals.
Keywords Histone deacetylase . Kilham rat virus . LEW1.WR1 rat . Type 1 diabetes . Inflammation
Introduction
Type 1 diabetes (T1D) is a proinflammatory progressive disease resulting in a specific destruction of insulin-producing islet beta cells [1]. Data from humans and animal models suggest that viruses may play a role in triggering T1D [2]; however the causal relationship between virus infections and disease development as well as mechanisms involved are unclear. We have used the LEW1.WR1 rat model of virus-induced T1D to better under- stand mechanisms in which viruses trigger T1D. Inoculating these animals with Kilham rat virus (KRV) results in T1D with disease characteristics similar to those of the human disease [3]. Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from histone, allowing histone molecules to wrap the DNA more tightly (reviewed in reference [4]). Inhibitors of HDAC have been found to suppress autoimmune inflammatory disorders, such as colitis [5], multiple sclerosis [6], and rheumatoid arthritis [7].
We recently demonstrated that KRV induces a robust proinflammatory response in the spleen and pancreatic lymph nodes from infected LEW1.WR1 rats via pathways linked with TLR9 pathways [8, 9]. We postulated that the innate immune system plays a key role in disease progression [10]. In this study, we examined whether and how the HDAC inhibitor ITF-2357 can prevent virus-induced T1D. We demonstrate that ITF-2357 down-modulated inflammation and prevented insulitis and T1D. Our data may imply that anti-inflammatory therapy with ITF-2357 may be an efficient therapeutic ap- proach for blocking virus-induced islet destruction.
Materials and methods
Animals, viruses, and cell lines
Specific pathogen-free LEW1.WR1 rats of both sexes were obtained from BRM Inc. (Worcester, MA). Animals were bred and housed in a specific pathogen-free facility and maintained in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996) and the guidelines of the Institutional Animal Care and Use Committee of the University of Colorado Denver. KRV was propagated and titered as previously described [11]. KRV and NRK cells used to produce KRV were obtained from stocks maintained in our laboratories [12]. The pancre- atic cell lines INS-1 was kindly provided by Dr. John Hutton (Barbara Davis Center for Childhood Diabetes, University of Colorado Denver).
Histologic staining
Pancreatic tissue was fixed for 24 h in 10 % neutral-buffered formalin, embedded in paraffin, cut (5–6 μm), and mounted on microscope slides. The tissue was stained with hematoxy- lin and eosin.
Virus-induced T1D, ITF-2357 therapy, blood, and lymphoid organ removal and in vitro cell activation
LEW1.WR1 rats at 21 days of age were injected i.p. with 1× 107 PFU of KRV as previously described [12]. Diabetes was diagnosed in infected rats with blood glucose level 250 mg/dl. ITF-2357 (from Italfarmaco, Milan, Italy) was administered orally at a dose of 30 μg/g body weight (a dose we found to be capable of inducing a 50 % reduction in the level of virus- induced IL-12 p40 in the serum) beginning on the day of KRV infection. Rats were administered with KRV only, KRV plus ITF-2357, or ITF-2357 only beginning on the day of virus infection on 5 or 14 consecutive as previously described [13]. The animals were monitored for insulitis and T1D for 40 days following infection. The treatment regimen used did not lead to any detectable adverse side effects. For Tand B cell analysis and quantitative RT-PCR analysis we used spleens from day- 5-infected rats. Spleen or INS-1 cells were cultured in the presence of medium alone or 2×106/ml PFU of KRV in the presence or absence of ITF-2357 for 18 and 4 h, respectively.
DNA purification and quantitative RT-PCR analysis for the detection of intestinal bacteria
Bacterial DNA was recovered using the QIAmp DNA stool mini kit (Qiagen, Valencia, CA) according to the manufac- turer’s instructions. The level of intestinal bacteria was deter- mined as previously described [21].
RNA extraction, cDNA synthesis, and quantitative RT-PCR
RNA extraction, cDNA synthesis, and quantitative RT-PCR were performed as previously described [21]. The primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). Their sequences have been previously pub- lished [21].
Flow cytometry
Cells were analyzed by flow cytometry as previously de- scribed [17]. A PerCP-conjugated anti-TCRα/β Ab (clone R73, mouse IgG1), a PE-conjugated anti-IL-2R α-chain Ab (CD25, clone OX-39, mouse IgG1), an APC-conjugated anti- CD4 Ab (clone OX-35, mouse IgG2a), a PerCP-conjugated anti-CD8α chain Ab (clone OX-8, mouse IgG1), a FITC- conjugated anti-CD45R Ab (a marker of B cells, clone HIS24, and mouse IgG2b), and appropriate isotype controls were purchased from BioLegend (San Diego, CA). An Efluor 450-conjugated mAb against Foxp3 (Clone FJK-16s, rat IGg2a) and fixation and permeabilization buffers were pur- chased from eBioscience (San Diego, CA). Flow cytometry was performed using a CYAN ADP instrument (Beckman Coulter), and the results were analyzed with FlowJo software.
KRV-specific cellular and humoral immunity and measurements of IL-12/IL-23 p40 and IL-10 levels
KRV-specific CD8+ IFN-γ+ and anti-KRV antibodies were detected in the spleen on day 12 following viral infection as previously described [14]. Anti-KRV Abs were detected in serum samples using a commercially available ELISA kit according to the manufacturer’s instructions (Alpha Diagnostic International, San Antonio, TX). The levels of IL-12 p40 and IL- 10 were quantitated using kits from Life Technologies according to the manufacturer’s instructions.
Statistical analysis
Statistical comparisons of diabetes-free survival among groups were performed using the method of Kaplan and Meier. Comparisons between more than two groups were performed with a one-way ANOVA with Bonferroni’s multiple compari- son test. Comparisons between two groups were performed with unpaired t test.
Results
ITF-2357 prevents insulitis and virus-induced T1D in the LEW1.WR1 rat
We examined the ability of ITF-2357 to prevent virus-induced T1D in the LEW1.WR1 rat model. LEW1.WR1 rats were injected with 1×107 PFU of KRV and administered ITF-2357 orally at a dose of 30 μg/g body weight on 14 or 5 consecutive days beginning on the day of viral inoculation. The results shown in Fig. 1a indicate that infection with KRV led to T1D in 57 % of the infected animals (n =49). In contrast, treating LEW1.WR1 rats with ITF-2357 for 14 days reduced the inci- dence of T1D to 17 % (n =12, p =0.02 vs. animals treated with KRVonly). Infection with KRVand ITF-2357 therapy on 5 days only led to T1D in 55 % of the treated rats (p >0.05 vs. KRV only).
Because we observed that ITF-2357 therapy prevented virus-induced T1D, we examined whether the difference in the incidence of T1D between ITF-2357-treated and untreated rats was linked to the degree of islet infiltration (n =6 per group; ≥20 islets per animal were screened for insulitis). We found that the majority of islets from prediabetic 21-day-infected animals showed insulitis whereas islets from rats that developed disease showed signs of destruction (Fig. 1b). In contrast, islets from animals injected with KRV and gavaged with ITF-2357 that were protected from diabetes were all free of insulitis at 40 days post-infection (Fig. 1b). Collectively, these data demonstrate that ITF-2357 protects from virus-induced insulitis and T1D.
ITF-2357 reverses KRV-induced accumulation of T and B cells in lymphoid organs
Infection of LEW1.WR1 rats with KRV results in a systemic inflammation evidenced by an increase in the T cell composi- tion of lymphoid organs on day 5 following infection [13]. We tested the hypothesis that ITF-2357-induced disease preven- tion may involve down-modulation of the T or B cell compart- ment in the spleen or pancreatic lymph nodes. We also were interested in defining whether the upregulation of Treg cells in the spleen or pancreatic lymph nodes is involved in disease prevention. To that end, animals were left untreated (n =3–30 per group), were infected with KRV (n =6–18 per group), were treated with KRV plus 30 μg/g body weight of ITF-2357 (n = 9–18 per group), or were administered with ITF-2357 only (n =3 per group) on 5 consecutive days beginning on the day of infection. Spleens and pancreatic lymph nodes were harvested and cells were analyzed by flow cytometry. The data presented in Fig. 2a indicate that treating LEW1.WR1 rats with KRV results in an increase and a reduction in the absolute number of splenic TCRαβ+ and Treg cells, respectively (p < 0.01 and p < 0.05, respectively, compared to uninfected). Treatment with ITF-2357, however, led to a reduction in the number of T cells (p <0.05 versus KRV only), but did not change the number of Treg cells. Consistent with these obser- vations, we found that ITF-2357 reversed the KRV-induced upregulation of T and B cells in the pancreatic lymph nodes. Collectively, the data raise the hypothesis that the mechanism in which ITF-2357 prevents T1D could be linked with down- modulation of systemic inflammation early in the course of T1D.
ITF-2357 down-modulates KRV-induced innate immunity in vitro and in vivo
We recently hypothesized that the mechanism of KRV-induced T1D involves the upregulation of proinflammatory pathways shortly following virus infection [8, 13, 14]. Because ITF-2357 was shown to possess anti-inflammatory activity [15], we tested the hypothesis that ITF-2357 halts T1D via down- regulating KRV-induced proinflammatory responses. To that end, we examined the effect of ITF-2357 administration on KRV-induced IL-12/IL-23 p40 and IL-10 in spleen cells in vitro (n =3–5 per group). Data presented in Fig. 3a demon- strate that incubating spleen cells from naïve rats in the pres- ence of KRV led to a substantial increase in the expression of the p40 subunit and IL-10 compared to uninfected rats (p < 0.001 for both cytokines). However, adding 1 μM of ITF-2357 to the cultures suppressed the expression of the p40 subunit and IL-10 compared to KRV only (p <0.001 for both cytokines). Adding ITF-2357 at a concentration of 0.1 μM to the cultures suppressed the expression of the p40 subunit (p <0.001 versus KRV only) but not that of IL-10. Lastly, 0.01 μM of ITF-2357 did not exert an inhibitory effect on the virus-induced p40 and IL-10 expression.
We recently observed that infection of the INS-1 beta tumor cell line with KRV in vitro results in the upregulation of transcripts for IRF-7, a transcription factor involved in type I interferon signaling pathways [16]. We tested the hypothesis that ITF-2357 could down-modulate IRF-7 gene expression in INS-1 beta cells. To that end, INS-1 beta cells were cultured in the presence or absence of KRV plus varying concentrations of ITF-2357 and the expression levels of transcripts for IRF-7 and KRV were assessed by quantitative RT-PCR analysis (n = 3–6). The presence of transcripts for KRV is indicative of virus infection and replication [17, 18]. Figure 3b demonstrates that KRV induced the expression of transcripts for IRF-7 and KRV in INS-1 cells (p <0.01 compared to uninfected). Adding 0.01 μM ITF-2357 to INS-1 cells cultured in the presence of KRV did not alter the level of IRF-7 gene expression. In contrast, incubating INS-1 cells in the presence of 1 μM of ITF-2357 led to a reduction in the level of IRF-7 transcripts (p <0.01 compared to KRV only). Lastly, similar levels of tran- scripts for KRV VP2 were detected in INS-1 cells cultured in the presence of KRV plus ITF-2357 at all ITF-2357 concen- trations tested compared to KRV only.
Because ITF-2357 suppressed KRV-induced innate immu- nity in vitro, we evaluated the effect of ITF-2357 on KRV- induced innate immunity in vivo. For this purpose, rats were treated with KRV plus ITF-2357 and the serum level of the p40 subunit of IL-12/IL-23 was measured on day 5 post- infection by ELISA (n =3–5 per group). The data presented in Fig. 3c demonstrate that animals administered with KRV only had a substantial increase in the level of the p40 subunit compared to uninfected rats (p <0.001). However, treating rats with KRV plus ITF-2357 on 5 consecutive days resulted in a twofold decrease in the level of the p40 subunit compared to KRV only (p <0.01). Treatment with ITF-2357 only did not alter the level of the p40 subunit compared to the uninfected control group.
Taken together, the data support the hypothesis that ITF- 2357 suppresses virus-induced proinflammatory responses in vitro in spleen and beta tumor cells and in vivo in the peripheral blood.
ITF-2357 modulates KRV-induced innate immunity in the spleen
In light of the observation that ITF-2357 suppressed KRV- induced innate immune responses in spleen cells vitro and in the peripheral blood in vivo, we addressed the possibility that ITF-2357 prevented T1D by targeting proinflammatory genes induced in the spleen on day 5 following virus infection [13]. We focused our analyses on genes that we previously identi- fied to be highly inducible shortly following infection [13, 14]. Groups of animals (n =5–7) were left untreated, were injected with KRVonly, were infected with KRVand gavaged with ITF-2357 beginning on the day of virus inoculation, or were treated with ITF-2357 only, and spleens were removed on day 5 post-infection. The data presented in Fig. 4 indicate that transcripts for KRV VP2 were detectable in the spleen from virus-infected animals but not in the tissue from control uninfected rats (p <0.01). Infection with KRV resulted in the upregulation of transcripts for IRF-7, CXCL-10 (p <0.05 and p <0.001, respectively) and the p40 subunit (p <0.01) com- pared to the control uninfected group. We further found that therapy with ITF-2357 did not alter the level of transcripts for KRV VP2, IRF-7, CXCL-10, the p40 subunit, IFN-γ, IL-1β, and STAT-1 compared to KRV only (Fig. 4 and data not shown). In contrast, treatment with KRV plus ITF-2357 resulted in the expression of higher levels of transcripts for IL-6 (p <0.05 compared to KRV only). Treatment with KRV plus ITF-2357 resulted in increased levels of IFN-γ compared to KRV only but this difference did not reach a statistically significant level. Finally, ITF-2357 did not alter the transcript level of IL-6, IRF-7, CXCL-10, the p40 subunit, IFN-γ, IL-1β, and STAT-1 in the pancreatic lymph nodes from KRV- plus ITF-2357-treated rats compared to KRVonly (data not shown). Overall, these data indicate that ITF-2357 does not exert a major effect on virus-induced gene expression in the spleen and further suggest that IL-6 may be a potential target of ITF- 2357.
ITF-2357 therapy does not interfere with the induction of KRV-specific adaptive immune responses and virus clearance
Because inhibitors of HDACs suppress innate immune re- sponses (this manuscript and references [19, 21]), we tested the effect of ITF-2357 on the ability of LEW1.WR1 rats to express virus-specific adaptive immune responses and clear the virus. To that end, we examined the effect of ITF-2357 on the induction of virus-specific CD8+IFN-γ+ T cells and the level of anti-KRV antibodies 12 days following virus infection in the spleen and serum, respectively. The effect of ITF-2357 on virus clearance was addressed by measuring the expression level of transcripts for KRV VP2 on days 5 and 40 in the spleen, pancreatic lymph nodes, and serum. Groups of rats (n =5–7) were left untreated, were injected with KRV only, were admin- istered with KRV plus ITF-2357, or were treated with ITF-2357 only. Figure 5a indicates that treating rats with KRV plus ITF- 2357 did not alter the frequency of CD8+IFN-γ+ cells com- pared to animals administered with KRVonly. Furthermore, rats treated with KRV plus ITF-2357 also had normal levels of virus-specific antibodies on days 12 and 40 (Fig. 5b, p >0.05 versus KRV only).
Consistent with the data indicating that LEW1.WR1 rats treated with ITF-2357 develop normal KRV-specific adaptive immune responses, data shown in Fig. 5c indicate that therapy with ITF-2357 may have altered the expression of KRV VP2 shortly after infection but overall did not compromise the ability of the rats to clear the virus from the spleen, pancreatic lymph nodes, and serum by day 40 post-infection. Taken together, these observations support the hypothesis that ITF-2357 does not interfere with the induction of KRV-induced adaptive immune responses or virus elimination from the host.
ITF-2357 reverses KRV-induced alterations in gut bacterial communities
We recently hypothesized that KRV-induced innate immunity influences the gut bacterial composition [21, 22]. Because we observed that ITF-2357 down-modulates inflammation, we were interested in defining whether it could reverse virus- induced modulation of the gut microbiome. To that end, animals were left untreated, administered with KRV with or without ITF-2357, or administered with ITF-2357 only (n =4– 6 per group). Fecal samples were collected on day 5, a time point at which alterations in the intestinal microbiota are detectable, and on day 12 when these differences are not detectable [21]. We evaluated the abundance of bacterial genera that we have recently linked with the course of virus- induced T1D [21, 22]. The data presented in Fig. 6a demon- strate that KRV increased abundance of the Bifidobacterium spp. and Clostridium spp. genera compared to uninfected animals (p <0.05 for both genera). However, administering animals with KRV plus ITF-2357 significantly reduced the abundance of both of these bacterial genera compared to KRV only (p <0.05 for both genera). On day 12 post-infection, the abundance of Clostridium spp. was significantly reduced in animals treated with KRV plus ITF-2357 or ITF-2357 only compared to KRV only (p <0.05), whereas the abundance of the other tested genera was comparable in treated versus untreated rats (Fig. 6b). These findings raise the hypothesis that ITF-2357-induced disease prevention correlates with re- storing healthier gut microbiota.
Discussion
The data from the present study show for the first time that ITF- 2357 can modulate virus-induced inflammatory responses in vitro and in vivo and prevent disease development without intervening with the induction of virus-specific adaptive im- mune responses or the ability of the host to clear the virus. We propose that targeting innate immune pathways with inhibitors of HDAC may prove to be an effective therapeutic approach for diabetes prevention.
We demonstrate that therapy with ITF-2357 reverses virus- induced systemic inflammation and restores normal T and B cell numbers in the spleen or pancreatic lymph nodes on day 5 post- infection [13]. We propose that blocking T and B cell accumu- lation in the spleen and pancreatic lymph nodes could lead toa reduction in the pool of potentially autoreactive T and B cells resulting in disease prevention. The observations suggesting that blocking KRV-induced innate immune responses has a benefi- cial effect on diabetes progression is consistent with a line of observations from both the BioBreeding Diabetes Resistant (BBDR) and the LEW1.WR1 rat models implicating early inflammation in disease mechanisms [13, 14]. For example, we demonstrated that activation of the innate immune system with TLR ligands promotes T1D in the BBDR and LEW1.WR1 rats [8, 23]. Furthermore, we found that down-modulation of virus-induced inflammation with steroids [13] or antibiotic ther- apy [21] prevented virus-induced islet destruction.
Our in vitro and in vivo data indicate that ITF-2357 sup- presses the KRV-induced expression of the p40 subunit and IL-10 and upregulates IL-6 expression in the spleen in vivo, potentially implicating these pathways in the mechanism of disease prevention. The anti-inflammatory effect of ITF-2357 observed in the LEW1.WR1 model is compatible with human data demonstrating that blood cells taken from ITF-2357- treated subjects shortly after drug infusion produce lower levels of proinflammatory cytokines previously implicated in islet destruction [24]. Our in vitro observations are reminis- cent of earlier reports demonstrating that inhibitors of HDAC can suppress the in vitro expression of IL-10 and IL-12 in monocytes or dendritic cells [19, 20, 25]. The interpretation of our in vitro versus in vivo data should be cautious since it could be that some of the in vivo effects of ITF-2357 may be mediated by drug metabolites present in vivo but not in vitro. In any case, similar to the ability of ITF-2357 to prevent T1D, HDAC inhibitors were also shown to suppress experimental colitis [5], multiple sclerosis [26], and arthritis [27, 28]. Furthermore, therapy with the HDAC inhibitor Trichostatin A protected the NOD mouse from islet destruction via mech- anisms linked with the upregulation of splenic CD4+CD62L+ cells, reduced insulitis, and restoration of normoglycemia and glucose-induced insulin secretion by beta cells [29]. Treating mice with ITF-2357 also protected from streptozotocin- induced disease by restoring normal serum nitrite levels and improving islet function [30].
The data show that ITF-2357 differentially affects innate immune responses in the serum versus spleen and pancreatic lymph nodes in vivo (this manuscript and data not shown). Indeed, we found that unlike the serum, ITF-2357 increased the IL-6 and IFN-γ gene expression in the spleen. In addition, ITF-2357 therapy led to a reduction in the level of the p40 subunit in the peripheral blood and KRV-activated spleen cells in vitro, but not spleen or pancreatic lymph nodes in vivo. The reason for these differences is unclear and could be linked with the amounts of ITF-2357 available in the microenviron- ment in vivo and the sensitivity of different cell types to the effects of ITF-2357.
The significance of the increase in IL-6 or IFN-γ gene expression to the mechanism of disease amelioration is unclear. IL-6 is a pleiotropic cytokine with both inflammatory and anti-inflammatory properties [31]. Thus, increased IL-6 expression could potentially lead to down-regulation of islet-specific adaptive immune responses and disease prevention [3, 10]. IFN-γ was previously associated with the prevention of T1D in the BioBreeding diabetes-prone rat model [32]. Our data from the spleen of LEW1.WR1 rats are reminiscent of earlier reports indicating that treating NOD mice with the HDAC inhibitor Trichostatin A led to increased expression of IFN-γ transcripts and protein in the spleen without altering the expression of inducible NO, IL-17, or TNF-α [29].
One potential mechanism whereby ITF-2357 could prevent islet destruction is by direct protection of beta cells from de- struction. This is supported by the observation that ITF-2357 suppressed the expression of KRV-induced IRF-7 in INS-1 cells in vitro. This observation is reminiscent of previous data show- ing that inhibition of HDAC with suberoylanilide hydroxamic acid (SAHA) and ITF-2357 resulted in lower levels of IL-1β- induced nitric oxide as well reduced cytokine-mediated decrease in insulin secretion in rat primary islet cells and the INS cell line [30, 33]. In addition, suppressing HDAC prevented cytokine- induced β-cell apoptosis and impaired β-cell function [34].
Our recent studies indicated that interplay between the in- nate immune system and intestinal bacteria may play a role in disease mechanisms [21]. The data from the present study show that ITF-2357 down-modulates inflammation and reverses KRV-induced alterations in the abundance of Bifidobacterium and Clostridium lend support to the hypothesis that inflamma- tion can result in altered intestinal microbiota. Whether ITF- 2357-induced modulation of the intestinal microbiome is di- rectly involved in the mechanism of disease amelioration re- mains to be seen.
In conclusion, our studies demonstrate that therapy with an inhibitor of HDAC can modulate virus-induced innate immune activation early in the course of T1D and prevent disease devel- opment. A better understanding of early proinflammatory path- ways involved in islet destruction could lead to the identification of new and effective therapeutic approaches to prevent islet destruction in genetically susceptible individuals.
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