Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice.

The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.

The role of αß T-cells in spontaneous regression of melanoma tumors in swine.

Using a porcine model, we describe Melanoma-Associated CD4+CD8hi T-lymphocytes (MATL) in peripheral blood that increase during melanoma regression. These MATL possess the CD4+CD8hi phenotype and they have their direct counterparts in Tumor Infiltrating Lymphocytes (TIL) isolated from melanoma loci. Both MATL and CD4+CD8hi TIL have a similar expression of selected markers indicating that they represent effector/memory αß T-cell subset. Moreover, although TIL also contain CD4-CD8+ T-cells, only CD4+CD8hi TIL expand during melanoma regression. Importantly, TIL isolated from different pigs and different melanoma loci among the same pig have similar composition of CD4/CD8 subsets, indicating that the composition of the MATL and TIL compartment is identical. Analysis of sorted cells from regressing pigs revealed a unique MATL subpopulation with mono-specific T-cell receptor that was further analyzed by sequencing. These results indicate that pigs regressing melanomas possess a characteristic population of recirculating T-cells playing a role in tumor control and regression.

The distribution of lymphoid cells in the small intestine of germ-free and conventional piglets.

Porcine ileum is populated with a high proportion of B cells but previous studies have shown that they are not developed there. While B cells prevail in the ileum even in germ-free animals, microbial colonization is a major factor that causes even a greater prevalence of B cells in the ileum and further differential representation of lymphoid cells throughout small intestine. Analysis of lymphoid subpopulations showed that the effector cells appear only after colonization. These include class-switched IgM(+)IgA(+) B cells, primed CD2(-)CD21(+) B cells, antibody-producing/memory CD2(+)CD21(-) B cells, and effector/memory CD4(+)CD8(+) αß Th cells. While colonization resulted in a uniform distribution of effector cells throughout the gut, it caused a decrease in the frequency of cytotoxic αß and CD2(+)CD8(+) γδ T cells. These results suggest that the ileum is a site where naive B cells expand presumably to increase antibody repertoire but the entire small intestine is immunofunctionally comparable.

B cell lymphogenesis in swine is located in the bone marrow.

A course and a site of B cell development in swine are not firmly known. In this study, we show that B cell lymphogenesis is located in the bone marrow (BM). According to expression of MHC class II (MHC-II), CD2, CD21, CD25, CD45RC, CD172a, swine workshop cluster (identification number) (SWC) 7, and µHC, porcine BM cells were resolved into seven subsets representing sequential stages of development. Profile of rearrangement-specific products and transcripts from sorted BM cells confirmed the proposed developmental pathway. The same developmental pathway was further proven by analysis of selection for productive rearrangements in Ig H chains and also by cultivation studies. Cultivation also showed that earliest precursors with incomplete DJ rearrangements can still revert their B cell differentiation and develop along myeloid lineage, whereas this is impossible for later developmental stages. Proliferation and the apoptotic potential of individual developmental stages as well as critical checkpoints were also identified. Colocalization experiments showed early colocalization of MHC-II/CD2/CD172a is replaced by colocalization of MHC-II/CD2/CD21/SWC7/IgM in immature cells, whereas CD25 and CD45RC did not colocalize with any other studied molecules. In this study, we also finally prove that the BM in pigs is fully functional in adult animals and that B lymphogenesis occurs there throughout life. To our knowledge, this is the first study showing a course and a direct site of B cell lymphogenesis in swine.

Porcine reproductive and respiratory syndrome (PRRS): an immune dysregulatory pandemic.

Porcine reproductive and respiratory disease syndrome (PRRS) is a viral pandemic that especially affects neonates within the "critical window" of immunological development. PRRS was recognized in 1987 and within a few years became pandemic causing an estimated yearly $600,000 economic loss in the USA with comparative losses in most other countries. The causative agent is a single-stranded, positive-sense enveloped arterivirus (PRRSV) that infects macrophages and plasmacytoid dendritic cells. Despite the discovery of PRRSV in 1991 and the publication of >2,000 articles, the control of PRRS is problematic. Despite the large volume of literature on this disease, the cellular and molecular mechanisms describing how PRRSV dysregulates the host immune system are poorly understood. We know that PRRSV suppresses innate immunity and causes abnormal B cell proliferation and repertoire development, often lymphopenia and thymic atrophy. The PRRSV genome is highly diverse, rapidly evolving but amenable to the generation of many mutants and chimeric viruses for experimental studies. PRRSV only replicates in swine which adds to the experimental difficulty since no inbred well-defined animal models are available. In this article, we summarize current knowledge and apply it toward developing a series of provocative and testable hypotheses to explain how PRRSV immunomodulates the porcine immune system with the goal of adding new perspectives on this disease.

Different anti-CD21 antibodies can be used to discriminate developmentally and functionally different subsets of B lymphocytes in circulation of pigs.

Monoclonal antibodies IAH-CC51, BB6-11C9.6 and B-Ly4 are routinely used to detect CD21 orthologue on the surface of porcine B lymphocytes. Cross-reactive studies show that IAH-CC51 and B-Ly4 recognize only a portion of B cells that are positive for pan-specific BB6-11C9.6. This indicates that CD21 is always present on all mature B cells but can be expressed in at least two differential forms, and these were assigned as CD21(a) and CD21(b). We used IAH-CC51 together with anti-CD2 to define four subpopulations of B cells. Ontogenetic and in vitro culture studies, analysis of cell size, expression of CD11b and class-switched phenotype together with measurement of proliferation and cell death, revealed that these subsets represent distinct populations. Phenotypic and functional features collectively suggest that CD21(b+) B cells are less mature than CD21(b-). The present work is the first to show that distinct subsets of mature B cells can express differential forms of CD21.

An activated immune and inflammatory response targets the pancreas of newborn pigs with cystic fibrosis.

BACKGROUND/AIMS: In cystic fibrosis (CF), pancreatic disease begins in utero and progresses over time to complete destruction of the organ. Although inflammatory cells have been detected in the pancreas of humans and pigs with CF, their subtypes have not been characterized. METHODS: Using four-color flow cytometry, we analyzed the surface antigens of leukocytes in pancreas, blood, and mesenteric lymph nodes (MLN) of newborn pigs with CF (CFTR(-/-) and CFTR(Δ)(F508/)(Δ)(F508)) and in those without CF (CFTR(+/-), CFTR(+/)(Δ)(F508), CFTR(+/+)). Pancreatic histopathology was examined with HE stain. RESULTS: CF pig pancreas had patchy distribution of inflammatory cells with neutrophils/macrophages in dilated acini, and lymphocytes in the interstitium compared to non-CF. B cells, effector (MHC-II(+)) and cytotoxic (CD2(+)CD8(+)) γδ T cells, activated (MHC-II(+) and/or CD25(+)) and effector (CD4(+)CD8(+)) αß T helper cells, effector natural killer cells (MHC-II(+)CD3(-)CD8(+)), and monocytes/macrophages and neutrophils were increased in the CF pig pancreas compared to pigs without CF. Blood and MLN leukocyte populations were not different between CF and non-CF pigs. CONCLUSIONS: We discovered an activated immune response that was specific to the pancreas of newborn CF pigs; inflammation was not systemic. The presence of both innate and adaptive immune cells suggests that the disease process is complex and extensive.

Ileal Peyer's patches are not necessary for systemic B cell development and maintenance and do not contribute significantly to the overall B cell pool in swine.

Based on studies of sheep, ileal Peyer's patches (IPP) have been regarded as a type of primary lymphoid tissue similar to the bursa of Fabricius in chicken. Because bursectomy results in B cell deficiency, we wondered whether resection of the IPP of piglets would have a similar effect. Comparison of IPP-resected, surgical shams and untreated germ-free piglets, all of which were later colonized with a defined commensal flora, demonstrated that resection of the IPP did not alter the level and phenotype of B and T cells in lymphoid tissues and the blood 10 wk after surgery. Additionally, colonization of IPP caused a shift from the fetal type of lymphocyte distribution to the adult type that is characterized by prevalence of B cells, with many of them representing IgA(+) switched B cells or displaying a more mature CD2(-)CD21(+) and CD2(-)CD21(-) phenotype. Moreover, colonization leads to appearance of effector CD4(+)CD8(+) αß T helper and CD2(+)CD8(-) γδ T cells. Comparison of germ-free with colonized pigs and experiments utilizing surgical transposition of jejunal Peyer's patch into terminal ileum or construction of isolated ileal loops indicated that lymphocyte development in IPP is dependent on colonization. Although our studies confirmed higher mitotic and apoptotic rates in IPP, they failed to identify any cell populations that resemble developing B lineage cells in the bone marrow. These results indicate that porcine IPP are not required for systemic B cell generation or maintenance, but they are secondary lymphoid tissue that appears important in immune responses to colonizing bacteria.

Antibody repertoire development in fetal and neonatal piglets. XX. B cell lymphogenesis is absent in the ileal Peyer's patches, their repertoire development is antigen dependent, and they are not required for B cell maintenance.

The continuous ileal Peyer's patches (IPP) of sheep are regarded as a type of mammalian bursal equivalent where B cells diversify their repertoire in an Ag-independent fashion. Anatomically and developmentally similar IPP occur in swine. Resection of ∼90% of the IPP in piglets at birth did not alter Ig levels in serum and secretions or retard diversification of the Ab repertoire when animals were maintained in isolators and colonized with a defined gut flora. Resection or sham surgery elevated IgG and IgA in serum and in lavage fluid from the gut, lung, and in saliva. No changes in the frequency of IgG-, IgA-, and IgM-containing cells in the spleen and peripheral lymph node were observed. Using an index that quantifies diversification of the VDJ repertoire, no differences were seen in three secondary lymphoid tissues between piglets lacking IPP and colonized controls, whereas both groups displayed >10-fold greater diversification than did late-term fetal piglets or piglets maintained germ-free. Somatic hypermutation was very low in fetal IPP and the IPP of germ-free piglets but increased 3- to 5-fold after colonization. D-J signal joint circles were not recovered in IPP, and V-DJ signal joint circles were 5-fold lower than in bone marrow and similar to those in thymus and spleen. We conclude that the porcine IPP are not a site of B cell lymphogenesis, do not undergo Ag-independent repertoire diversification, and are not primary lymphoid tissue since they are not required for maintenance of Ig levels in serum and secretions.

The ontogeny of the porcine immune system.

Cellular and humoral aspects of the immune response develop sequentially in the fetus. During the ontogeny, the pluripotent stem cells emerge and differentiate into all hematopoietic lineages. Basic questions including the identification of the first lympho-hematopoietic sites, the origin of T and B lymphocytes, the development of different subpopulations of alphabeta T, gammadelta T and B lymphocytes as well as development of innate immunity and the acquisition of full immunological capacities are discussed here for swine and compared with other species. The description of related topics such as fertilization, morphogenesis, maternal-fetal-neonatal physiology and early neonatal development are also discussed.

Constitutive expression of IL-18 and IL-18R in differentiated IEC-6 cells: effect of TNF-alpha and IFN-gamma treatment.

The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, brush border enzyme activities, and gene expression of apoptotic markers. However, the addition of IL-18 stimulated production and release of the chemokine IL-8. These data suggest that IEC-6 cells may be not only a source of IL-18 but also a target for its action.

The isolator piglet: a model for studying the development of adaptive immunity.

The period from late gestation to weaning in neonatal mammals is a critical window when the adaptive immune system develops and replaces the protection temporarily provided by passive immunity and pre-adaptive antibodies. It is also when oral tolerance to dietary antigen and the distinction between commensal and pathogenic gut bacteria becomes established resulting in immune homeostasis. The reproductive biology of swine provides a unique model for distinguishing the effects of different factors on immune development during this critical period because all extrinsic factors are controlled by the experimenter. This chapter reviews this early stage of development and the usefulness of the piglet model for understanding events during this transitional stage. The review also describes the major features of the porcine immune system and the immune stimulatory and dysregulatory factors that act during this period. The value of the model to medical science in such areas as food allergy, organ transplantation, cystic fibrosis and the production of humanized antibodies for immuno-therapy is discussed.

Antibody repertoire development in fetal and neonatal piglets: XIX. Undiversified B cells with hydrophobic HCDR3s preferentially proliferate in the porcine reproductive and respiratory syndrome.

Porcine respiratory and reproductive syndrome virus (PRRSV) causes an extraordinary increase in the proportion of B cells resulting in lymphoid hyperplasia, hypergammaglobulinemia, and autoimmunity in neonatal piglets. Spectratypic analysis of B cells from neonatal isolator piglets show a non-Gaussian pattern with preferential expansion of clones bearing certain H chain third complementary region (HCDR3) lengths. However, only in PRRSV-infected isolator piglets was nearly the identical spectratype observed for all lymphoid tissues. This result suggests dissemination of the same dominant B cell clones throughout the body. B cell expansion in PRRS was not associated with preferential VH gene usage or repertoire diversification and these cells appeared to bear a naive phenotype. The B cell population observed during infection comprised those with hydrophobic HCDR3s, especially sequences encoded by reading frame 3 of DHA that generates the AMVLV motif. Thus, the hydropathicity profile of B cells after infection was skewed to favor those with hydrophobic binding sites, whereas the normally dominant region of the hydropathicity profile containing neutral HCDR3s was absent. We believe that the hypergammaglobulinemia results from the products of these cells. We speculate that PRRSV infection generates a product that engages the BCR of naive B cells, displaying the AMVLV and similar motifs in HCDR3 and resulting in their T-independent proliferation without repertoire diversification.

B cell development and VDJ rearrangement in the fetal pig.

Hematopoietic activity of the swine has been documented in three phases during fetal ontogeny. The hematopoietic system develops first in the yolk sac, then in fetal liver and finally in the bone marrow. Using flow cytometry (FCM) and molecular biological techniques we show that B-cell lymphogenesis and the appearance of B cells follows a pattern. First, VDJ rearrangement occurs at the 20th day of gestation (DG20) in the yolk sac at a time when light chain transcription is absent. Next, B-cell lymphogenesis is detected at DG30 in the fetal liver. Thereafter, bone marrow becomes the major B lymphopoietic organ (DG45). In yolk sac and fetal liver, more than 90% of the VDJ rearrangements were in-frame but expression of micro heavy chain could not be clearly detected by FCM. However, cells with a putative phenotype of B-cell precursors are present. These cells express high levels of MHC class II (SLA-DR) and low levels of CD2 and CD25. CDR3 length analysis (spectratyping) indicates that the heavy chain repertoire is oligoclonal at this time with large inter-animal variations. Consistent with our earlier reports, fetal VDJ rearrangements are not mutated and there is no evidence for an age-dependent increase in TdT activity or a change in V(H) and D(H) usage from those used by B-cells formed in the yolk sac or fetal liver. However, our findings indicate major differences in the regulatory environment and/or selective pressures in yolk sac and fetal liver versus bone marrow. In contrast with the yolk sac and fetal liver, the proportion of in-frame VDJ rearrangements in the bone marrow correspond to a value indicative of random recombination.