Rotaviruses (RV) are a major cause of severe dehydrating diarrhea in infants and young children worldwide. Micronutrient deficiencies, intestinal dysbiosis, and RV genetic variability contribute to reduced human RV (HRV) vaccine efficacy and increased RV diarrhea burden in low income settings. Here we discuss the advantages of the gnotobiotic (Gn) pig model to study HRV pathogenesis, immunity, and vaccines and factors that influence them. Using the Gn pig model, we recently demonstrated that probiotics alone, or together with lactogenic immune factors modulate neonatal immune responses to virulent and attenuated HRV. Additionally, we showed that the Gn pig is an excellent model to study probiotic effects at the molecular level using transcriptome profiling. Further, prenatal vitamin A deficiency (VAD) in neonatal Gn pigs profoundly modulated immune responses, exacerbating HRV infection and compromising HRV vaccine efficacy. Thus, high VAD prevalence in children in developing countries may aggravate HRV disease and compromise HRV vaccine efficacy.
Chapter 2.7
Gnotobiotic Neonatal Pig Model of Rotavirus Infection and Disease
A.N. Vlasova
S. Kandasamy
L.J. Saif Food Animal Health Research Program, The Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, United States
Abstract
Keywords
gnotobiotic pig
neonatal pig
rotavirus
probiotics
commensal bacteria
micronutrients
vitamin A deficiency
1. Introduction
Poor sanitation and water treatment systems, and lack of medical care and efficacious vaccines translate into nearly 1,600,000 deaths among children <5 years old and several million hospitalizations due to severe, dehydrating diarrhea annually (Tagbo et al., 2014). Rotavirus (RV) has been recognized as a major cause of severe diarrhea in infants and young children, associated with ∼453,000 deaths worldwide annually (232,000 deaths in sub-Saharan Africa) (Tate et al., 2012). Besides supportive care and fluid/electrolyte replacement therapy, no specific antiviral treatments are available.
Rotaviruses belong to a genus within the Reoviridae family. They possess a genome of 11 segments of double-stranded RNA, encoding 6 structural viral proteins (VPs) and 6 nonstructural proteins (NSPs). The glycoprotein VP7 (G type) and the hemagglutinin VP4 (P type) independently induce neutralizing antibodies (Abs) (Estes and Kapikan, 2007).
Two RV vaccines are currently licenced in many countries: Rotarix (GlaxoSmithKline), a monovalent vaccine of sero-/genotype G1P1A[8] and RotaTeq (Merck), a pentavalent vaccine containing genes encoding human RV (HRV) VP7 proteins of serotypes G1, G2, G3, and G4 and genotype P1A[8] on the G6P7[5] bovine RV genetic background (WC3 strain) (Vesikari et al., 2006). Although, these vaccines effectively prevent severe RV gastroenteritis in developed countries (>80%), they show reduced efficacy (∼30–50%) in impoverished countries, where RV diarrhea is most severe (Gray, 2011). The reduced efficacy of oral vaccines and increased mortality rates from RV diarrhea in children are often associated with their poor macro- and micronutrient status (specifically vitamin A deficiency, VAD) and/or intestinal dysbiosis. Vaccine efficacy may be further affected by the significant genetic diversity of RV strains. In developed countries, direct and indirect medical costs related to RV disease are estimated to be €400 million in Europe and over $1 billion in the United States (Desselberger and Huppertz, 2011). Therefore, the need for animal experimentation to study HRV pathogenesis, vaccine-induced immunity and possible interventions is paramount.
A number of laboratory and agricultural animal models (reviewed in Desselberger and Huppertz, 2011) have been developed to study the mechanisms of RV pathogenesis and immunity, including piglets, rabbits, rats, lambs, and calves. However, the majority of the mechanistic studies have been conducted with the adult mouse model established in 1990 (Ward et al., 1990). Although of great convenience, mouse models have significant limitations because of their homogenous and inbred genetic background, physiology, anatomy, and immunity, all of which are distinct from those of humans. Additionally, a major limitation of the mouse models is that they do not support the efficient replication of human RVs. Also mice only have a short-term (up to 15 days of life) susceptibility to diarrhea induced by most murine RVs (Franco and Greenberg, 1999, 2000). Consequently, the murine (as well as rabbit) models reproduce only HRV infection but not disease; while studies using lamb and calf models were done only with homologous (ovine and bovine, respectively) RVs.
Further, several investigators have reported that oral inoculation of different nonhuman primates with either cell culture-adapted simian (SA11) or HRVs (including strain Wa) resulted in diarrheal illness during the first week of life (Kalter et al., 1983; Leong and Awang, 1990; Mitchell et al., 1977; Petschow et al., 1992; Wyatt et al., 1976). However, no illness, virus shedding or seroconversions were observed in the older animals to evaluate active immunity postchallenge. Additionally, the transplacental transfer of maternal Abs together with the economic and ethical issues associated with the use of primate models greatly decreases their value and convenience.
The use of swine in biomedical research has been widely accepted because pigs and humans share numerous similarities in anatomy, physiology, metabolism, feeding patterns, and immunity (Meurens et al., 2012) (Table 2.7.1). Apart from the primate and murine immune systems, the porcine immune system is the best characterized, offering a wide range of established research protocols and tools (Summerfield, 2009). Additionally, as outbred animals, pigs are more representative of human population heterogeneity than inbred mouse strains. Piglets are born immunologically immature and devoid of circulating maternal Abs because the sow’s placenta (epitheliochorial type) blocks the transfer in utero of immunoglobulins (Hammerberg et al., 1989; Friess et al., 1981). Neonatal pigs resemble infants in their physiology, anatomy and in the development of mucosal immunity (Butler et al., 2007; Yuan et al., 1996; Yuan and Saif, 2002). Gnotobiotic (Gn) pigs are a unique animal model to assess HRV disease pathogenesis and to evaluate RV vaccines in the presence or absence of commensal gut microbiota. HRV-infected Gn pigs exhibit diarrhea, transient viremia, and intestinal lesions (villous atrophy) mimicking those in children (Azevedo et al., 2005; Ward et al., 1996). The Gn pig is susceptible to HRV diarrhea for at least 8 weeks of age, which is the time necessary to assess disease pathogenesis and protective immunity (Azevedo et al., 2004). The correlates of protection against challenge with HRVs are serum and intestinal RV-specific IgA Abs but not the serum levels of RV-neutralizing Abs (predominantly IgG) (Saif et al., 1996; Azevedo et al., 2004). However, VP7- or VP4-specific Abs protect against challenge with homologous or heterologous RVs (Yuan and Saif, 2002; Saif et al., 1996; Hoshino et al., 1988; Yuan et al., 1996); whereas, VP6-specific IgA Abs do not (Saif et al., 1996; Yuan et al., 2001). Further, being omnivores, pigs represent a translatable model for human research to study the effects of macro- and micronutrient deficiencies and supplementation on HRV pathogenesis and immunity via dietary manipulations (Zijlstra et al., 1997; Kandasamy et al., 2014a; Vlasova et al., 2013a; Chattha et al., 2013a). Finally, the complete pig genome sequence has provided an important resource for transcriptomic research to understand the host-microbiota-pathogen interactions at the molecular level (Groenen et al., 2012; Wernersson et al., 2005). Thus, the Gn pig model represents a unique system and may serve as a preclinical pipeline to study the various aspects of interactions among the immunologically immature neonatal host, dietary components, lactogenic immune factors, commensal/probiotic bacteria and HRV under strictly controlled manipulative conditions.
Table 2.7.1
Advantages of Porcine, Murine, and Nonhuman Primate Models for Biomedical Research
| Advantages of the porcine model for biomedical research | Murine models | Nonhuman primate models |
| Availability (most important meat-producing livestock species worldwide) | Yes | No |
| Size similar to human infant | No | Yes |
| Possibility of performing analogous surgical procedures and of collecting many samples | No | Yes |
| Similar anatomy | No | Yes |
| Omnivorous (similar gastrointestinal physiology) | No | Yes |
| Closely resemble humans for >80% of immune parameters analyzed (Dawson et al., 2013) | No (<10%) | Yes |
| Cheaper and ethically more acceptable than nonhuman primates | Yes | N/A |
| Various breeds (541), outbred and inbred | Yes | Yes/No |
| Large litter size (10–12 piglets/litter) | Yes | No |
| Standardized breeding conditions | Yes | No |
| High pig genome and protein sequence homologies with human counterparts (up to 95%) (Wernersson et al., 2005) | No | Yes |
| Prolonged susceptibility (up to 8 weeks of life) to some human pathogens, including HRVs | No | No |
1.1. Lactobacilli and Bifidobacteria Modulate Innate and Adaptive Immune Responses to Human Rotavirus Infection in Neonatal Gnotobiotic Pigs
Intestinal commensals regulate development of gut immunity (Macpherson and Harris, 2004) and influence the outcome of viral infections (Wilks and Golovkina, 2012) including RV infections (Uchiyama et al., 2014) (Fig. 2.7.1). An understanding of the complex interplay among host immunity, intestinal commensals/probiotics and viral pathogens will provide novel strategies to prevent enteric viral infections including RV. Enhancing the efficacy and effectiveness of RV vaccines in impoverished countries is essential for prevention of the disease.
Figure 2.7.1 (A) Probiotic-associated immune-mediators/immune mechanisms, and (B) interactions between probiotics and the immune system modelled in mice and Gn pigs.
(A) In the intestinal lumen, probiotics (1) inhibit certain viruses directly by producing lactic acid, H2O2, bacteriocins, and other inhibitory agents; (2) probiotics can also preserve the integrity of the epithelium and compete with pathogens for intestinal epithelial cell (IEC) receptors; (3) Lactobacilli could also capture viruses by lectin-mediated binding to viral glycoproteins and in this way prevent infection; (4) Lactobacilli/Bifidobacteria enhance the local immune system during health and disease and thereby inhibit infection; (5) nitric oxide (NO) produced by Lactobacilli plays a role in microbicidal and tumoricidal activities and in immunopathology; (6) Bifidobacteria-derived short chain fatty acids (SCFA) have immunomodulatory effects: inhibiting dendritic cell development, decreasing IL-12 levels, but increasing LI-23 production by DCs, and inducing Fas-mediated T cell apoptosis. (B) In homeostasis, intestinal epithelial cells (IECs) secrete mucins and AMPs in response to the commensal microbiota, regulating microbial replication, and interaction with intestinal mucosa. Additionally, IECs produce BAFF and APRIL factors, stimulating activated B (plasma) cells that produce secretory IgA (sIgA) in the lumen that further limits microbial interaction with the epithelium. Under homeostatic conditions, commensal microbiota stimulate the secretion of cytokines [including thymus stimulating lymphoprotein (TSLP), IL-33, IL-23, IL-25, and TGFβ] by IECs that promote development of antigen presenting cells [macrophages (Mφ) and dendritic cells (DCs)]. Antigen presenting cells induce regulatory T (Treg) cell generation through TGFβ- and retinoic acid (RA)-dependent mechanisms. Through APC and Treg derived TGFβ and IL-10, the antiinflammatory nature of the intestine is maintained by inhibiting or reducing effector responses. Intestinal innate lymphoid cells (ILCs), including natural killer (NK) cells, lymphoid tissue inducer (LTi) cells, and γδ T cells, produce IL-22 that regulates expression of tight and adherent junction (TJ and AJ) proteins by IECs, regulating intestinal barrier function. Upon pathogen invasion, mucosal injury, or dysbiosis, microbe associated molecular patterns (MAMPs) stimulate the secretion of proinflammatory and pluripotent cytokines by IECs (including, IL-6, IL-1, and IL-18) and APCs (including IL-6, IL-23, and IL-12) that induce effector CD4+ T cells (Th1, Th2, and Th17) via IL-23 or IL-12 signalling leading to generation of pathogen specific IgA+ B cells and diverse interactions with intestinal ILCs. Intestinal innate lymphoid cells respond to proinflammatory cytokines to upregulate IL-22, which helps to maintain the epithelial barrier, and IL-17A and IL-17F, which are involved in neutrophil recruitment and inflammatory responses. (AMP, antimicrobial peptides; BAFF, B-cell activating factor; APRIL, A proliferation-inducing ligand).
(A) In the intestinal lumen, probiotics (1) inhibit certain viruses directly by producing lactic acid, H2O2, bacteriocins, and other inhibitory agents; (2) probiotics can also preserve the integrity of the epithelium and compete with pathogens for intestinal epithelial cell (IEC) receptors; (3) Lactobacilli could also capture viruses by lectin-mediated binding to viral glycoproteins and in this way prevent infection; (4) Lactobacilli/Bifidobacteria enhance the local immune system during health and disease and thereby inhibit infection; (5) nitric oxide (NO) produced by Lactobacilli plays a role in microbicidal and tumoricidal activities and in immunopathology; (6) Bifidobacteria-derived short chain fatty acids (SCFA) have immunomodulatory effects: inhibiting dendritic cell development, decreasing IL-12 levels, but increasing LI-23 production by DCs, and inducing Fas-mediated T cell apoptosis. (B) In homeostasis, intestinal epithelial cells (IECs) secrete mucins and AMPs in response to the commensal microbiota, regulating microbial replication, and interaction with intestinal mucosa. Additionally, IECs produce BAFF and APRIL factors, stimulating activated B (plasma) cells that produce secretory IgA (sIgA) in the lumen that further limits microbial interaction with the epithelium. Under homeostatic conditions, commensal microbiota stimulate the secretion of cytokines [including thymus stimulating lymphoprotein (TSLP), IL-33, IL-23, IL-25, and TGFβ] by IECs that promote development of antigen presenting cells [macrophages (Mφ) and dendritic cells (DCs)]. Antigen presenting cells induce regulatory T (Treg) cell generation through TGFβ- and retinoic acid (RA)-dependent mechanisms. Through APC and Treg derived TGFβ and IL-10, the antiinflammatory nature of the intestine is maintained by inhibiting or reducing effector responses. Intestinal innate lymphoid cells (ILCs), including natural killer (NK) cells, lymphoid tissue inducer (LTi) cells, and γδ T cells, produce IL-22 that regulates expression of tight and adherent junction (TJ and AJ) proteins by IECs, regulating intestinal barrier function. Upon pathogen invasion, mucosal injury, or dysbiosis, microbe associated molecular patterns (MAMPs) stimulate the secretion of proinflammatory and pluripotent cytokines by IECs (including, IL-6, IL-1, and IL-18) and APCs (including IL-6, IL-23, and IL-12) that induce effector CD4+ T cells (Th1, Th2, and Th17) via IL-23 or IL-12 signalling leading to generation of pathogen specific IgA+ B cells and diverse interactions with intestinal ILCs. Intestinal innate lymphoid cells respond to proinflammatory cytokines to upregulate IL-22, which helps to maintain the epithelial barrier, and IL-17A and IL-17F, which are involved in neutrophil recruitment and inflammatory responses. (AMP, antimicrobial peptides; BAFF, B-cell activating factor; APRIL, A proliferation-inducing ligand).
In our initial experiments, we focused on delineating the immunomodulatory effects of commonly used probiotics, such as Lactobacilli and Bifidobacteria on immune responses to RV. Lactobacillus spp. are the components of normal intestinal microflora of humans and pigs (Ahrne et al., 2005), and Lactobacilli and Bifidobacteria are the two major phyla in naturally delivered and breastfed infants. Also a recent study showed that the presence of certain commensal bacterial species was negatively associated with the development of adaptive immunity to oral poliovirus vaccine (Huda et al., 2014).
1.1.1. Interactions Among Probiotics, HRV and Innate Immunity (Summarized in Table 2.7.2)
Innate immunity provides the initial defense against pathogens (see Chapter 2.8), but little is known about the impact of probiotics on innate immune responses to HRV infection or vaccines. Dendritic cells (DCs) and macrophages play major roles in initiating innate immunity. Dendritic cells are a heterogeneous group of potent antigen-presenting cells (APC) that express specialized pattern recognition receptors including various antigen uptake receptors, such as Fc receptors and certain C-type lectins (Kelsall et al., 2002). They have a strong capacity to prime T-cell responses and regulate B-cell proliferation and isotype switching (Hivroz et al., 2012; Balazs et al., 2002; Johansson et al., 2000; Wykes and MacPherson, 2000). Two major subsets of DCs were characterized in various species including humans, mice, and swine (Summerfield et al., 2003): (1) conventional (cDCs) that have a major function of antigen presenting and (2) plasmacytoid (pDCs) DCs also known as natural interferon alpha producers. The latter cells were shown to critically influence immune responses to RV (Mesa et al., 2007; Deal et al., 2013). Further, DCs and other innate immune cells express Toll-like receptors (TLRs), a family of pattern recognition receptors, which are central to activation of innate immunity (Medzhitov, 2001). Among the various TLRs, TLR2 (expressed on cell surface) in combination with TLR1/TLR6 recognizes gram positive bacterial components, such as peptidoglycans, lipoteichoic acids, and lipoproteins (Song and Lee, 2012). Intracellular TLR3 recognizes double-stranded (ds)RNA of microbes and intracellular TLR9 recognizes unmethlylated CpG motifs that are commonly found in microbial genomes (Song and Lee, 2012).
Recently, we assessed the impact of Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on innate and adaptive (see Section 1.1.2) immunity in RV vaccinated piglets (Vlasova et al., 2013b) (Table 2.7.2). Dual colonization with LGG and Bb12 resulted in less severe diarrhea and reduced virus shedding titers compared to uncolonized piglets and differentially modulated mucosal and systemic innate immunity during HRV infection of Gn pigs (Vlasova et al., 2013b). These probiotics exerted inhibitory effects on DC populations at the systemic level as evident by lower frequencies of activated splenic conventional and plasmacytoid DC (cDC and pDC) in probiotic colonized vaccinated piglets compared to uncolonized vaccinated piglets post-HRV challenge. Alternatively, this finding may also be a result of the reduced HRV replication in the probiotic colonized piglets. Consistent with reduced HRV shedding/diarrhea, LGG + Bb12 colonized piglets had lower IFNα responses compared to uncolonized piglets post-HRV challenge. However, postchallenge, probiotic colonized, vaccinated piglets had higher frequencies of activated pDCs/cDCs in ileum and blood compared to uncolonized vaccinated piglets. We observed a synergistic interaction between the attenuated HRV (AttHRV) vaccine and LGG and Bb12 colonization as evident by increased frequencies of ileal TLR9+ mononuclear cells (MNCs) in intestinal tissues of probiotic colonized, RV vaccinated piglets compared to uncolonized AttHRV vaccinated piglets prechallenge. The probiotics alone had little or no long-term stimulatory effect on frequencies of TLR9 + MNCs. Further, the increased TLR9+ MNC frequencies prechallenge coincided with a higher protective effect against virus shedding and diarrhea observed postvirulent HRV challenge. In contrast, the LGG and Bb12 colonized, vaccinated piglets had decreased frequencies of ileal TLR2+ and TLR4+ MNCs compared to uncolonized vaccinated piglets. An earlier study of adult human subjects reported increased TLR2 and TLR4 expression in submucosal immune cells of inflamed intestinal mucosa compared to healthy mucosa (Hausmann et al., 2002). Thus, regulating the expression of specific TLRs by these probiotics in the small intestine might play a role in intestinal immune homeostasis and also prevent excessive inflammatory responses during viral infection.
Table 2.7.2
Summary of Probiotic (Lactobacillus spp. and Bifidobacterium lactis Bb12) Effects on Various Immune Parameters, and Responses to AttHRV Vaccine and Virulent HRV Infection Studied in Gn Pigs.
| Probiotic/probiotic combination | Commensal microbiota | AttHRV vaccine | VirHRV infection/challengea | Observed effects of the probiotics | References |
| LGG + Bb12 | No No | Yes No | Yes Yes | Postchallenge, clinical parameters: decreased severity of HRV infection and disease; postchallenge, innate immune parameters: decreased systemic, but promoted intestinal innate immune responses and immune trafficking, differentially affected TLR responses (decreased pro-inflammatory, increased B-cell promoting); postchallenge, adaptive immune responses: promoted adaptive immune (including B, effector and regulatory T cell) responses | Vlasova et al. (2013b), Kandasamy et al. (2014b), Chattha et al. (2013b) |
| LA + LR | No | No | Yes | Innate immune parameters: differentially affected APC frequencies in HRV infected and noninfected piglets, increased TLR expression by blood cDCs; Adaptive immune responses: promoted T cell responses and decreased pro-inflammatory cytokine production | Zhang et al. (2008a), Zhang et al. (2008c), Wen et al. (2009), Azevedo et al. (2012), Wen et al. (2011) |
| LGG | No | No | Yes | Clinical parameters: decreased severity of HRV infection and disease; Innate immune parameters: decreased intestinal damage and other effects of HRV infection | Liu et al. (2013), Wu et al. (2013) |
| LA | No | Yes | No | Adaptive immune responses: increased adaptive B and T cell immune responses | Zhang et al. (2008b) |
| LGG | Yes | No | Yes | Moderated HRV effects on intestinal microbiota | Zhang et al. (2014) |
a In the experiments that involved VirHRV infection/challenge, the immune/clinical parameters were assessed after the VirHRV infection/challenge.
Investigators have reported that immunomodulatory effects vary with strain (Medina et al., 2007) and composition of the probiotic bacteria (Gackowska et al., 2006). Thus, we also assessed the impact of two other lactic acid producing probiotic bacteria (LAB), Lactobacillus acidophilus (LA) and Lactobacillus reuteri (LR), on intestinal and systemic innate immune responses (Zhang et al., 2008a,b,c; Wen et al., 2009). Compared to uninfected negative control piglets, HRV infection alone significantly increased monocytes/macrophages, but not the cDC population in ileum. However, LA + LR colonized HRV infected piglets had lower frequencies of monocytes/macrophages compared to HRV only infected piglets in ileum. Additionally, probiotic colonized piglets had lower frequency of activated macrophages post-HRV infection. The antigen presenting cell (APC) populations in spleen were significantly reduced in LA + LR colonized, compared to uncolonized piglets, postvirulent HRV infection. Previous studies by others showed that LR reduced TNFα production in lipopolysaccharide (LPS) treated macrophages (Pena et al., 2004) and suppressed proinflammatory cytokines in macrophages from children with Crohn’s disease (Lin et al., 2008). Similarly, colonization of piglets with LA + LR significantly reduced TNFα cytokine secreting cells in ileum and spleen post-HRV challenge (Azevedo et al., 2012). Thus, reduction in total, as well as activated intestinal monocyte/macrophage populations, and decreased inflammatory cytokine production in LAB colonized piglets during HRV infection indicates that these probiotics have a protective effect on inflammatory damage during HRV infection.
Colonization of Gn piglets with LAB alone resulted in significant modulation of innate immunity. LA + LR dual colonization significantly increased both monocytes/macrophages and cDC populations in ileum in comparison to uncolonized Gn piglets (Zhang et al., 2008c). Further, in the absence of HRV infection, probiotic colonization alone increased the frequencies of TLR2 and TLR9 positive cDC in blood (Wen et al., 2009). These results indicate that LA and LR alone had significant stimulatory effects on the innate immune system.
Intestinal epithelial cells are the target cells for RV, and their anatomic location facilitates interactions with probiotics and intestinal commensals. In a recent study, LGG colonization modulated HRV effects on the levels of tight junction and adherent junction proteins (Liu et al., 2013) and downregulated autophagy in ileal epithelium after HRV infection (Wu et al., 2013). Thus, it appears that probiotics can alleviate the RV induced pathological changes in intestinal epithelial cells and supplementation of probiotics might be a potential strategy to reduce the severe consequences of RV infection on intestinal epithelial cells.
1.1.2. Functional Effects of Probiotics on Adaptive Immunity to RV (Table 2.7.2)
Virus specific B-cell responses play an important role in clearing RV infection and are critical for development of antiviral T-cell responses implicated in controlling primary viral infections (Franco and Greenberg, 1995). Further, a significant correlation was also observed between virus specific serum IgA Ab levels and RV vaccine efficacy in children (Patel et al., 2013). Since RV vaccines lack efficacy in impoverished countries, where diarrhea mortality is highest, we have focused our studies on enhancing the immunogenicity and protective efficacy of RV vaccines using economical approaches, such as probiotics and intestinal commensals.
Dual colonization of LGG and Bb12 probiotics had significant effects on HRV vaccine induced B- and T-cell responses. B-cell responses, including activation of intestinal B cells and RV specific IgA Ab titers were enhanced in vaccinated, probiotic colonized piglets compared to uncolonized, vaccinated piglets postvirulent HRV challenge (Kandasamy et al., 2014b). Further, T-cell responses, specifically ileal T regulatory cells, and systemic IFNγ producing T-cell responses, were increased in probiotic colonized, vaccinated compared to uncolonized vaccinated piglets (Chattha et al., 2013b). Importantly, the probiotic induced immunomodulatory effects on adaptive immune responses coincided with decreased diarrhea severity and reduced fecal virus shedding.
Similar to LGG and Bb12 effects on B-cell responses, LA probiotic significantly enhanced the immunogenicity of AttHRV vaccine responses as indicated by higher numbers of ileal HRV specific IgA and IgG antibody secreting cells (ASCs) and increased intestinal IFNγ producing T cells compared to uncolonized piglets post HRV inoculation (Zhang et al., 2008b). Apart from individual effects of LA on adaptive vaccine-specific immunity, dual-colonization of LA and LR significantly modulated the types of γδ T-cell responses (critical for early responses to infections at epithelial surfaces) during HRV infection of Gn piglets without vaccination (Wen et al., 2011). There were lower numbers of inflammatory type CD2 + CD8 − γδ T cells and higher regulatory type CD2 + CD8+ γδ T cells (Saalmuller et al., 1990) in LA + LR probiotic colonized piglets in comparison to uncolonized piglets postvirulent HRV infection. Additionally, higher systemic IFNγ and IL4 cytokine responses in LA + LR colonized compared to uncolonized HRV infected piglets suggest that LAB modulated both Th1 and Th2 immunity, respectively (Wen et al., 2009). Thus, the probiotics tested had measurable beneficial effects on AttHRV vaccine protective efficacy and immunogenicity, and they moderated the severity of HRV diarrhea when given at least 21 days prior to HRV challenge (Chattha et al., 2013b). However, whether these observed beneficial effects can be reproduced by these probiotics in the presence of complex microbiota has yet to be determined.
1.1.3. Neonatal Pig Models Colonized With Complex Intestinal Microbiota of Human or Swine Origin
The Gn piglet model can also be utilized to address important questions of how intestinal microbiota of human infants modulate or are modulated by host immunity, intestinal health, and RV infections (Pang et al., 2007; Wen et al., 2014). Initial studies using humanized piglets, that is, piglets transplanted with intestinal microbiota from a breast-fed infant (delivered by Caesarean section), revealed that RV infection shifted bacterial abundance from phylum Fimicutes to phylum Proteobacteria (Zhang et al., 2014), whereas LGG supplementation prevented the HRV infection-induced changes in the microbial community. By contrast, our preliminary data demonstrate that in Gn pigs colonized with defined microbiota of swine origin, HRV infection increased the relative abundance of phyla Firmicutes and Bacteroidetes and decreased that of Proteobacteria in the large and small intestine (Saif, Rajashekara, Vlasova et al., unpublished). Consistent with our findings, alterations in the composition of the Bacteroides phylum after RV infection were observed in human subjects (Zhang et al., 2009), suggesting that the composition of intestinal microbiota also influences the outcome of RV infections. Finally, in an ongoing study, we are evaluating the interactions among intestinal microflora, malnutrition, and RV infection to identify efficacious dietary interventions using Gn piglets transplanted with fecal microbiota from a healthy vaginally delivered breast-fed infant (Saif, Vlasova, Rajashekara et al., unpublished). Thus, humanized piglets or piglets colonized with other complex defined microbiota are a valuable model to study the effects of diet, RV infection and vaccines on microbial ecology and host immunity.
1.2. Interactions Between Lactogenic Immune Factors, Probiotics, Neonatal Immune System and Human Rotavirus Vaccine in a Gnotobiotic Pig Model
There are few studies on the impact of selected probiotics on responses to oral vaccines in neonates in the context of colostrum/milk (col/milk) feeding. We have recently examined how LGG + Bb12 colonization with or without col/milk (to mimic breastfed versus formula-fed infants) affects development of B cell responses to an oral AttHRV vaccine in the relevant Gn pig model.
In agreement with previous findings that breast-milk promotes growth of Bifidobacteria and Lactobacilli, supplementation of col/milk (naturally containing TGFβ and other growth factors) in our study increased fecal probiotic shedding suggesting that milk containing regulatory cytokines (such as TGFβ) and other soluble factors (glycans etc.) can promote establishment and extended colonization by probiotics (LGG + Bb12) (Ahrne et al., 2005; Yoshioka et al., 1983; Rinne et al., 2005). Breast milk is a major source of TGFβ for neonates when intrinsic production is limited (Penttila, 2010; Nguyen et al., 2007) promoting intestinal immune responses, including class-switch to IgA, induction of regulatory T lymphocytes, attenuation of proinflammatory responses, and reduction of immune-mediated and allergic conditions (Kalliomaki et al., 1999). Unexpectedly, an increase in probiotic fecal shedding was not observed in col/milk fed vaccinated pigs, possibly due to the AttHRV vaccine related reduction in serum TGFβ and increase in proinflammatory and Th1 cytokines (IL6 and IL12), resulting in conditions less favorable for commensal colonization (Azevedo et al., 2006).
Lower counts of probiotics detected in cecum/colon of col/milk fed pigs, irrespective of vaccination, suggested a differential impact of col/milk on fecal bacterial shedding versus intestinal distribution or mucosal adherence. Maternal Abs in sow col/milk to bacterial components including peptidoglycan may prevent mucosal adhesion of probiotics resulting in lower mucosa-associated bacterial counts as observed in suckling Gn mice previously (Kramer and Cebra, 1995). Another study demonstrated that in humans, bacteria from B. animalis subsp. lactis taxon are rarely found in intestinal biopsy samples (mucosa-adherent), whereas they are frequently present in fecal samples (luminal), suggesting that this taxon may not be an abundant component of the mucosa-adherent bacteria in humans or pigs (Turroni et al., 2009).
Combined probiotic colonization and col/milk supplementation in vaccinated pigs enhanced serum IgA HRV Ab titers and intestinal IgA HRV ASC levels, which was not observed in vaccinated pigs that did not receive col/milk, suggesting complex interactions between probiotics and col/milk components. Col/milk containing HRV Abs transiently suppressed serum IgA Ab responses after two vaccine doses irrespective of probiotic colonization, but this effect was ameliorated after the three dose vaccine regimen. Thus, colonization with LGG + Bb12 in breast fed vaccinated infants (with preexisting maternal HRV Abs) may overcome the suppressive effects of maternal Abs at least on IgA Ab responses. Similar to our study, Isolauri et al. (1995) showed enhanced RV IgA Ab responses in LGG fed infants of unknown breastfeeding status after oral immunization with live oral RV vaccine. In contrast, supplementation of Bifidobacterium breve strain Yakult (BBG-01) in breast fed children resulted in no significant difference in vibriocidal Ab responders following oral cholera vaccination (Matsuda et al., 2011). The quantity of maternal Abs received in utero (humans) or through colostrum (pigs) and the duration of breast milk feeding are critical factors in determining active Ab responses of neonates to oral vaccines.
Probiotics alone did not enhance IgA Ab responses to oral HRV vaccine in noncol/milk supplemented pigs, suggesting lack of adjuvancy of LGG + Bb12 for primary IgA Ab responses under the conditions tested. Similarly, Perez et al. (2010) have shown a lack of adjuvant effect of probiotics in enhancement of tetanus and pneumococcal IgA and IgG Abs in children, but this was after parenteral immunizations. In contrast, use of Bifiobacterium spp. in formula fed infants enhanced poliovirus and RV IgA Abs to the respective vaccines (Holscher et al., 2012; Mullie et al., 2004). Probiotic effects vary with strain, dose, vaccine type and route, level of maternal Abs, duration of breast feeding, age, the existing microflora, and other factors. Further studies are needed to elucidate reasons for inconsistent results.
Probiotic colonization resulted in significantly lower serum HRV IgG Ab titers and IgG HRV ASC in HRV vaccinated Gn pigs that did not receive col/milk. This suggests that LGG + Bb12 may enhance gut barrier integrity in multiple ways, similar to other probiotics (Ohland and Macnaughton, 2010). This would reduce systemic translocation and exposure to AttHRV and reduce IgG HRV ASC and Ab responses. However, this hypothesis was not investigated directly in our study. Thus, our results using the Gn pig model suggest that feeding LGG + Bb12 in breastfed infants may be advantageous, not only by directly enhancing IgA HRV Abs, but also by reducing systemic exposure and preventing adverse clinical effects of HRV gastroenteritis.
1.3. Gut Transcriptome Responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in Neonatal Gnotobiotic Piglets
Molecular mechanisms of probiotic action on neonatal intestinal mucosal immunity are largely undefined. Using a transcriptomic approach, we assessed mucosal tissue responses to LGG or LA monocolonization in comparison to uncolonized Gn piglets (Kumar et al., 2014). Results suggest that transcriptomic responses vary with the strain of probiotic, duration of probiotic colonization and region of the intestinal tract. Immediately after probiotic colonization (day 1), both LA and LGG induced higher transcriptional responses in ileum, whereas at later stages (7 days), LGG, but not LA, induced profound changes in expression of transcripts in duodenum. Both of these probiotics seem to polarize mucosal immunity toward Th1 type as indicated by higher expression of chemokine (C–C motif) ligand 9 [CCL9; macrophage inflammatory protein-1 gamma (MIP-1γ)] in LA and LGG piglets and higher granzyme (serine proteases involved in apoptosis) expression in LA piglets.
Compared to LA, LGG significantly modulated genes associated with the following pathways: inflammatory response, immune cell trafficking, and hematological system development in the duodenum. Pathways associated with immune modulation and carbohydrate metabolism were highly altered by LGG, whereas LA predominantly induced changes in energy and lipid metabolism-related trancriptomic responses. Further LA, but not LGG, induced prominent changes in transcription of vitamin A related genes in duodenum. Thus, LA and LGG differentially modulated major pathways in intestinal tissues. Further, both LGG and LA colonization resulted in higher expression of glucagon-like peptide 2 receptor (GLP2R) which regulates villus height and crypt depth in the small intestine (Jeppesen et al., 2001). LGG colonization also increased expression of claudin-8, a tight junction protein that regulates paracellular permeability (Ulluwishewa et al., 2011). Collectively, our intestinal tissue transcriptomic study revealed that Lactobacilli have prominent impacts on the host immune and metabolic functions. Our future studies will assess the effect of commonly used probiotics on transcriptomic responses of individual cell types in the small intestine. These studies may illuminate the precise mechanisms of probiotic action on mucosal immunity and suggest strategies to tailor preventive and therapeutic probiotic therapy for RV infections.
1.4. Prenatal Vitamin A Deficiency Alters Immune Responses to Virulent Human Rotavirus/Human Rotavirus Vaccines in a Gnotobiotic Pig Model
Vitamin A deficiency (VAD) in children is a significant health concern in impoverished countries. Even marginal (subclinical) VAD may compromise various aspects of innate and adaptive immune responses, resulting in enhanced susceptibility to infectious diseases (Fig. 2.7.2). Prophylactic supplemental vitamin A recommended by WHO has reduced the incidence of diarrhea associated morbidity and mortality in these countries (Humphrey et al., 1996; Imdad et al., 2011). To investigate interactions among VAD, supplemental vitamin A, AttHRV vaccine and virulent HRV infection, we have established a VAD Gn pig model with significantly decreased liver vitamin A levels and positive serum 30-day dose-response test (Ferraz et al., 2004) (Fig. 2.7.3). We showed that VAD increases the severity of HRV-induced diarrhea and impairs innate, mucosal, and systemic adaptive immune responses to HRV infection and AttHRV vaccines. Two separate trials using G1P1A[8] HRV Wa (genetically similar to commercial monovalent HRV vaccine, Rotarix) and pentavalent RotaTeq (Merck) yielded similar results indicating that VAD compromised the protective efficacy of both vaccines (Kandasamy et al., 2014a; Chattha et al., 2013a). HRV challenge resulted in decreased vitamin A levels in serum early postchallenge as was previously shown for measles virus (West, 2000), suggesting that HRV-induced intestinal villous atrophy (Ward et al., 1996) may affect vitamin A metabolism or its absorption from the milk diet.
Figure 2.7.2 Vitamin A (Retinoic Acid) and VAD effects on the immune system.
Retinol is taken up from the blood and oxidized to retinal by retinol dehydrogenases (RDH) and then to all-trans-retinoic acid (RA) by retinal dehydrogenases (RALDH), expressed predominantly by dendritic cells (DCs). The evidence generated in various animal models (rodent and avian species mostly) indicates that RA affects most major immune cell subsets including natural killer (NK) cells, innate lymphoid cells (ILC), T cells [Th1, Th2, Th17, and regulatory (Treg)], B cells; and it regulates Ig and cytokine production by the immune cells. Additionally, RA affects the cell cycle by decreasing lymphocyte (mononuclear cell) proliferation rates and increasing lymphocyte differentiation. Studies in rodent, avian, and porcine models demonstrated that VAD has multiple and varied effects on the immune system, including increased lymphocyte proliferation rates and decreased apoptosis that result in systemic expansion of various immune cells of lymphoid and myeloid origin (immature or incompletely differentiated), dysregulated cytokine responses and decreased IgA, CD103+ DC, Treg and toll-like receptor (TLR) 3 responses.
Retinol is taken up from the blood and oxidized to retinal by retinol dehydrogenases (RDH) and then to all-trans-retinoic acid (RA) by retinal dehydrogenases (RALDH), expressed predominantly by dendritic cells (DCs). The evidence generated in various animal models (rodent and avian species mostly) indicates that RA affects most major immune cell subsets including natural killer (NK) cells, innate lymphoid cells (ILC), T cells [Th1, Th2, Th17, and regulatory (Treg)], B cells; and it regulates Ig and cytokine production by the immune cells. Additionally, RA affects the cell cycle by decreasing lymphocyte (mononuclear cell) proliferation rates and increasing lymphocyte differentiation. Studies in rodent, avian, and porcine models demonstrated that VAD has multiple and varied effects on the immune system, including increased lymphocyte proliferation rates and decreased apoptosis that result in systemic expansion of various immune cells of lymphoid and myeloid origin (immature or incompletely differentiated), dysregulated cytokine responses and decreased IgA, CD103+ DC, Treg and toll-like receptor (TLR) 3 responses.
Figure 2.7.3 Prenatal VAD effects on the immune responses in gnotobiotic (Gn) pig model.
Prenatal VAD in Gn piglets resulted in increased severity of human rotavirus (HRV) disease and infection, dysregulated [increased total dendritic cell (DC), but decreased CD103+ DC frequencies, increased early IFNα responses, followed by a significant decrease in MNC capacity to produce IFNα in response to HRV restimulation in vitro, etc.] innate immune responses and decreased adaptive [including IgA and regulatory T (Treg) cell] immune responses. Vitamin A supplementation at the treatment concentrations and times tested did not reverse the VAD effects. Retinoic acid is required for lymphoid tissue inducer (LTi) cell development in the prenatal period; thus, its lack or insufficiency prenatally may result in impaired LTi cell function and the associated permanent or long-term deficiency of the innate and adaptive immune responses.
Prenatal VAD in Gn piglets resulted in increased severity of human rotavirus (HRV) disease and infection, dysregulated [increased total dendritic cell (DC), but decreased CD103+ DC frequencies, increased early IFNα responses, followed by a significant decrease in MNC capacity to produce IFNα in response to HRV restimulation in vitro, etc.] innate immune responses and decreased adaptive [including IgA and regulatory T (Treg) cell] immune responses. Vitamin A supplementation at the treatment concentrations and times tested did not reverse the VAD effects. Retinoic acid is required for lymphoid tissue inducer (LTi) cell development in the prenatal period; thus, its lack or insufficiency prenatally may result in impaired LTi cell function and the associated permanent or long-term deficiency of the innate and adaptive immune responses.
We showed that porcine retinol binding protein 4 (RBP4) and retinoic acid receptor-alpha (RARα) mRNAs (reflective of respective protein levels) are expressed by splenic MNCs and that their levels are affected by VAD, vitamin A supplementation and HRV challenge. Vlasova et al. (2013a) VAD decreased RARα mRNA levels and lowered relative responses to supplemental vitamin A. This suggests that in VAD pigs, supplemental vitamin A may not be metabolized to all-trans retinoic acid (ATRA) efficiently, or insufficient RARα expression results in aberrant cell signalling and the observed lack of consistent compensatory effects of vitamin A supplementation.
1.4.1. VAD Effects on the Innate Immune Responses
Numerous studies have demonstrated that VAD decreases resistance to and aggravates respiratory and enteric infections by depressing immune function. In our experiments, we observed not just a decrease, but VAD-induced dysregulation of IFNα production in response to AttHRV vaccine or HRV infection. Significantly higher levels of circulating IFNα in VAD piglets early postchallenge were consistent with higher HRV replication titers. However, later postchallenge, the capacity for IFNα production by MNCs from VAD piglets ex vivo was significantly decreased as compared to prechallenge and to MNCs from vitamin A sufficient (VAS) piglets postchallenge. This, together with higher amounts of IFNα in VAD piglets’ sera, suggests that imbalanced IFNα release by immune cells may contribute to increased inflammation and does not efficiently control HRV infection. Although RVs are known to be potent IFNα inducers, the role of IFNα in RV clearance is uncertain and varies between homologous and heterologous infections (Feng et al., 2008). Moreover, type I and II interferons were not demonstrated to be major inhibitors of RV replication in mice (Angel et al., 1999).
VAD also resulted in higher total frequencies of pDCs (gut tissues) and cDCs (all tissues) prechallenge in agreement with previous findings by others that VAD causes a systemic expansion of myeloid cells in sensitive to skin carcinogenesis (SENCAR) mice and an increase in different lymphocytes/lymphoid DCs in C57BL/6J mice (Duriancik et al., 2010; Kuwata et al., 2000).
Small intestinal CD103+ DCs are imprinted with an ability to metabolize vitamin A (retinol) and generate gut-tropic T cells (expressing CC chemokine receptor-9 and α4β7). Significantly lower CD103+ pDC and cDC frequencies in ileum, duodenum, and spleen of VAD piglets compared to VAS piglets suggest that DC-associated vitamin A metabolism was altered in VAD piglets. The loss of CD103 integrin by colonic DCs during experimentally induced colitis was described in mice (Strauch et al., 2010), suggesting that VAD-induced MNC necrosis could contribute to, or be the result of the significantly decreased CD103+ DC frequencies. Ultimately, disruption of signaling between DCs and T/B-lymphocytes may be due to lack of proper antigen presentation to the latter cells, resulting in the lower HRV specific IgA Ab titers that we observed (Kandasamy et al., 2014a; Chattha et al., 2013a) in VAD piglets (see later). Finally, increased frequencies of apoptotic MNCs in VAS piglets prechallenge confirm that adequate vitamin A levels control apoptosis, a mechanism involved in regulation of autoimmunity and T-cell tolerance (Jin et al., 2010).
1.4.2. VAD Effects on the Adaptive Immune Responses
Our Gn pig VAD model demonstrated that reduced protection against HRV challenge coincided with significantly higher CD8 T-cell frequencies in the blood and intestinal tissues, higher proinflammatory (IL12) and 2–3-fold lower antiinflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Similar to our study, lower frequencies of CD4 T cells and increased frequencies of CD8 T cells in intestinal tissues and spleen have been reported in VAD mouse and rat models (Duriancik et al., 2010; Bjersing et al., 2002). The AttHRV vaccinated VAD pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions (Chattha et al., 2013a). Furthermore, regulatory T-cell responses were compromised in VAD pigs (Chattha et al., 2013a).
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