Sukumar Kandasamy

Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model.

Rotaviruses (RVs) are a leading cause of childhood diarrhea. Current oral vaccines are not effective in impoverished countries where the vaccine is needed most. Therefore, alternative affordable strategies are urgently needed. Probiotics can alleviate diarrhea in children and enhance specific systemic and mucosal Ab responses, but the T cell responses are undefined. In this study, we elucidated the T cell and cytokine responses to attenuated human RV (AttHRV) and virulent human RV (HRV) in gnotobiotic pigs colonized with probiotics (Lactobacillus rhamnosus strain GG [LGG] and Bifidobacterium lactis Bb12 [Bb12]), mimicking gut commensals in breastfed infants. Neonatal gnotobiotic pigs are the only animal model susceptible to HRV diarrhea. Probiotic colonized and nonvaccinated (Probiotic) pigs had lower diarrhea and reduced virus shedding postchallenge compared with noncolonized and nonvaccinated pigs (Control). Higher protection in the Probiotic group coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-β and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization in vaccinated pigs enhanced innate serum IFN-α, splenic and circulatory IFN-γ–producing T cells, and serum Th1 cytokines, but reduced serum Th2 cytokines compared with noncolonized vaccinated pigs (Vac). Thus, LGG+Bb12 induced systemic Th1 immunostimulatory effects on oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge in Vac+Pro compared with Vac pigs. Previously unreported intestinal CD8 Tregs were induced in vaccinated groups postchallenge. Thus, probiotics LGG+Bb12 exert divergent immunomodulating effects, with enhanced Th1 responses to oral AttHRV vaccine, whereas inducing Treg responses to virulent HRV.

Lactobacilli and Bifidobacteria Promote Immune Homeostasis by Modulating Innate Immune Responses to Human Rotavirus in Neonatal Gnotobiotic Pigs

The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.

Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model

B cells play a key role in generation of protective immunity against rotavirus infection, a major cause of gastroenteritis in children. Current RV vaccines are less effective in developing countries compared to developed countries. Commensals/probiotics influence mucosal immunity, but the role of early gut colonizing bacteria in modulating intestinal B cell responses to RV vaccines is largely unknown. We co-colonized neonatal gnotobiotic pigs, the only animal model susceptible to HRV diarrhea, with 2 dominant bacterial species present in the gut of breastfed infants, Lactobacillus rhamnosus strain GG and Bifidobacterium animalis lactis Bb12 to evaluate their impact on B cell responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine. Following HRV challenge, probiotic-colonized, AttHRV vaccinated piglets had significantly lower fecal scores and reduced HRV shedding titers compared to uncolonized, AttHRV vaccinated pigs. The reduction in HRV diarrhea was significantly correlated with higher intestinal IgA HRV antibody titers and intestinal HRV-specific IgA antibody secreting cell (ASC) numbers in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs. The significantly higher small intestinal HRV IgA antibody responses coincided with higher IL-6, IL-10 and APRIL responses of ileal mononuclear cells (MNCs) and the immunomodulatory effects of probiotics genomic DNA on TGF-β and IL-10 responses. However, serum RV IgG antibody titers and total IgG titers were significantly lower in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs, both pre- and post-challenge. In summary, LGG and Bb12 beneficially modulated intestinal B cell responses to HRV vaccine.

Prenatally Acquired Vitamin A Deficiency Alters Innate Immune Responses to Human Rotavirus in a Gnotobiotic Pig Model

We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in a gnotobiotic (Gn) piglet model of HRV diarrhea. The VAD and vitamin A–sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000 IU) and challenged with virulent HRV (VirHRV). Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD piglets prechallenge, but decreased substantially postchallenge as compared with VAS pigs. We observed significantly higher frequency of CD103 (integrin αEβ7) expressing DCs in VAS versus VAD piglets postchallenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post-VirHRV challenge, we observed longer and more pronounced diarrhea and higher VirHRV fecal titers in nonvaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFN-α levels were induced in control VAD versus VAS piglet sera at postchallenge day 2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs prechallenge also produced more IFN-α. In contrast, at postchallenge day 10, we observed reduced IFN-α levels in VAD pigs that coincided with decreased TLR3+ MNC frequencies. Numbers of necrotic MNCs were higher in VAD pigs in spleen (coincident with splenomegaly in other VAD animals) prechallenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) postchallenge. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.

Comparison of probiotic lactobacilli and bifidobacteria effects, immune responses and rotavirus vaccines and infection in different host species.

Different probiotic strains of Lactobacillus and Bifidobacterium genera possess significant and widely acknowledged health-promoting and immunomodulatory properties. They also provide an affordable means for prevention and treatment of various infectious, allergic and inflammatory conditions as demonstrated in numerous human and animal studies. Despite the ample evidence of protective effects of these probiotics against rotavirus (RV) infection and disease, the precise immune mechanisms of this protection remain largely undefined, because of limited mechanistic research possible in humans and investigated in the majority of animal models. Additionally, while most human clinical probiotic trials are well-standardized using the same strains, uniform dosages, regimens of the probiotic treatments and similar host age, animal studies often lack standardization, have variable experimental designs, and non-uniform and sometime limited selection of experimental variables or observational parameters. This review presents selected data on different probiotic strains of lactobacilli and bifidobacteria and summarizes the knowledge of their immunomodulatory properties and the associated protection against RV disease in diverse host species including neonates.

Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity.

Rotavirus (RV) causes significant morbidity and mortality in children worldwide. The intestinal microbiota plays an important role in modulating host–pathogen interactions, but little is known about the impact of commonly used probiotics on human RV (HRV) infection. In this study, we compared the immunomodulatory effects of Gram-positive (Lactobacillus rhamnosus strain GG [LGG]) and Gram-negative (Escherichia coli Nissle [EcN]) probiotic bacteria on virulent human rotavirus (VirHRV) infection and immunity using neonatal gnotobiotic piglets. Gnotobiotic piglets were colonized with EcN, LGG, or EcN+LGG or uncolonized and challenged with VirHRV. Mean peak virus shedding titers and mean cumulative fecal scores were significantly lower in EcN-colonized compared with LGG-colonized or uncolonized piglets. Reduced viral shedding titers were correlated with significantly reduced small intestinal HRV IgA Ab responses in EcN-colonized compared with uncolonized piglets post-VirHRV challenge. However the total IgA levels post-VirHRV challenge in the intestine and pre-VirHRV challenge in serum were significantly higher in EcN-colonized than in LGG-colonized piglets. In vitro treatment of mononuclear cells with these probiotics demonstrated that EcN, but not LGG, induced IL-6, IL-10, and IgA, with the latter partially dependent on IL-10. However, addition of exogenous recombinant porcine IL-10 + IL-6 to mononuclear cells cocultured with LGG significantly enhanced IgA responses. The greater effectiveness of EcN in moderating HRV infection may also be explained by the binding of EcN but not LGG to Wa HRV particles or HRV 2/4/6 virus-like particles but not 2/6 virus-like particles. Results suggest that EcN and LGG differentially modulate RV infection and B cell responses.

Probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine

Breast milk (colostrum [col]/milk) components and gut commensals play important roles in neonatal immune maturation, establishment of gut homeostasis and immune responses to enteric pathogens and oral vaccines. We investigated the impact of colonization by probiotics, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) with/without col/milk (mimicking breast/formula fed infants) on B lymphocyte responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine in a neonatal gnotobiotic pig model. Col/milk did not affect probiotic colonization in AttHRV vaccinated pigs. However, unvaccinated pigs fed col/milk shed higher numbers of probiotic bacteria in feces than non-col/milk fed colonized controls. In AttHRV vaccinated pigs, col/milk feeding with probiotic treatment resulted in higher mean serum IgA HRV antibody titers and intestinal IgA antibody secreting cell (ASC) numbers compared to col/milk fed, non-colonized vaccinated pigs. In vaccinated pigs without col/milk, probiotic colonization did not affect IgA HRV antibody titers, but serum IgG HRV antibody titers and gut IgG ASC numbers were lower, suggesting that certain probiotics differentially impact HRV vaccine responses. Our findings suggest that col/milk components (soluble mediators) affect initial probiotic colonization, and together, they modulate neonatal antibody responses to oral AttHRV vaccine in complex ways.

Prenatal vitamin A deficiency impairs adaptive immune responses to pentavalent rotavirus vaccine (RotaTeq®) in a neonatal gnotobiotic pig model.

Vitamin A deficiency (VAD) is associated with increased childhood mortality and morbidity in impoverished Asian and African countries, but the impact of VAD on rotavirus (RV) vaccine or infection is poorly understood. We assessed effects of gestational and dietary induced pre- and post-natal VAD and vitamin A supplementation on immune responses to a pentavalent rotavirus vaccine, RotaTeq(®) in a neonatal gnotobiotic pig model. Vaccine efficacy was assessed against virulent G1P[8] human rotavirus (HRV) challenge. VAD and vitamin A sufficient (VAS) piglets were derived from dietary VAD and VAS sows, respectively. VAD piglets had significantly lower levels of hepatic vitamin A compared to that of VAS piglets. RotaTeq(®)-vaccinated VAD piglets had 350-fold higher fecal virus shedding titers compared to vaccinated VAS piglets post-challenge. Only 25% of vaccinated non-vitamin A supplemented VAD piglets were protected against diarrhea compared with 100% protection rate in vaccinated non-supplemented VAS piglets post-challenge. Intestinal HRV specific immune responses were compromised in VAD piglets. Vaccinated VAD piglets had significantly lower ileal HRV IgG antibody secreting cell (ASC) responses (pre-challenge) and duodenal HRV IgA ASC responses (post-challenge) compared to vaccinated VAS piglets. Also, intestinal HRV IgA antibody titers were 11-fold lower in vaccinated VAD compared to vaccinated VAS piglets post-challenge. Persistently elevated levels of IL-8, a pro-inflammatory mediator, and lower IL-10 responses (anti-inflammatory) in vaccinated VAD compared to VAS piglets suggest more severe inflammatory responses in VAD piglets post-challenge. Moreover higher IFN-γ responses pre-challenge were observed in VAD compared to VAS piglets. The impaired vaccine-specific intestinal antibody responses and decreased immunoregulatory cytokine responses coincided with reduced protective efficacy of the RV vaccine against virulent HRV challenge in VAD piglets. In conclusion, VAD impaired antibody responses to RotaTeq(®) and vaccine efficacy. Oral supplementation of 100,000 IU vitamin A concurrent with RV vaccine failed to increase the vaccine efficacy in VAD piglets.

Escherichia coli Nissle 1917 protects gnotobiotic pigs against human rotavirus by modulating pDC and NK-cell responses.

Lactobacillus rhamnosus GG (LGG), a gram‐positive lactic acid bacterium, is one of the most widely used probiotics; while fewer gram‐negative probiotics including Escherichia coli Nissle 1917 (EcN) are characterized. A mechanistic understanding of their individual and interactive effects on human rotavirus (HRV) and immunity is lacking. In this study, noncolonized, EcN‐, LGG‐, and EcN + LGG‐colonized neonatal gnotobiotic (Gn) pigs were challenged with HRV. EcN colonization is associated with a greater protection against HRV, and induces the highest frequencies of plasmacytoid dendritic cells (pDCs), significantly increased NK‐cell function and decreased frequencies of apoptotic and TLR4+ mononuclear cells (MNCs). Consistent with the highest NK‐cell activity, splenic CD172+ MNCs (DC enriched fraction) of EcN‐colonized pigs produced the highest levels of IL‐12 in vitro. LGG colonization has little effect on the above parameters, which are intermediate in EcN + LGG‐colonized pigs, suggesting that probiotics modulate each others effects. Additionally, in vitro EcN‐treated splenic or intestinal MNCs produce higher levels of innate, immunoregulatory and immunostimulatory cytokines, IFN‐α, IL‐12, and IL‐10, compared to MNCs of pigs treated with LGG. These results indicate that the EcN‐mediated greater protection against HRV is associated with potent stimulation of the innate immune system and activation of the DC‐IL‐12‐NK immune axis.

In vivo gut transcriptome responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in neonatal gnotobiotic piglets.

Probiotics facilitate mucosal repair and maintain gut homeostasis. They are often used in adjunct with rehydration or antibiotic therapy in enteric infections. Lactobacillus spp have been tested in infants for the prevention or treatment of various enteric conditions. However, to aid in rational strain selection for specific treatments, comprehensive studies are required to delineate and compare the specific molecules and pathways involved in a less complex but biologically relevant model (gnotobiotic pigs). Here we elucidated Lactobacillus rhamnosus (LGG) and L. acidophilus (LA) specific effects on gut transcriptome responses in a neonatal gnotobiotic (Gn) pig model to simulate responses in newly colonized infants. Whole genome microarray, followed by biological pathway reconstruction, was used to investigate the host-microbe interactions in duodenum and ileum at early (day 1) and later stages (day 7) of colonization. Both LA and LGG modulated common responses related to host metabolism, gut integrity, and immunity, as well as responses unique to each strain in Gn pigs. Our data indicated that probiotic establishment and beneficial effects in the host are guided by: (1) down-regulation or upregulation of immune function-related genes in the early and later stages of colonization, respectively, and (2) alternations in metabolism of small molecules (vitamins and/or minerals) and macromolecules (carbohydrates, proteins, and lipids). Pathways related to immune modulation and carbohydrate metabolism were more affected by LGG, whereas energy and lipid metabolism-related transcriptome responses were prominently modulated by LA. These findings imply that identification of probiotic strain-specific gut responses could facilitate the rational design of probiotic-based interventions to moderate specific enteric conditions.