Human Innate Immune Response Research Articles

Current cellular innate immune hurdles in pig-to-primate xenotransplantation

The generation of pigs lacking Galalpha1,3Gal (alphaGal) or overexpressing human regulators of complement has largely overcome the barrier of hyperacute xenograft rejection. Nevertheless, delayed xenograft rejection remains a major hurdle, including humoral responses against nonalphaGal epitopes, and cellular innate immune responses. This review addresses the early interactions between the porcine endothelium and human natural killer cells, neutrophils, and monocytes/macrophages. Whether human leukocyte recruitment to, and lysis of, porcine endothelial cells includes direct recognition of alphaGal remains a matter of debate. Although the human natural killer receptor natural killerRP1A may directly recognize alphaGal, several studies did not reveal significant differences regarding adhesion, transmigration, and cytotoxicity using porcine endothelial cells expressing or lacking alphaGal. The strong human monocyte response against pig cells partly relies on the inability of porcine ligands to ligate inhibitory human receptors, such as SIRPalpha. Strategies to overcome cellular innate immune responses include transgenic expression in porcine cells of human ligands for inhibitory receptors, together with species-specific blocking of porcine ligands mediating human leukocyte interactions. Cellular human innate immune responses are increasingly recognized as barriers to successful pig-to-human xenotransplantation, and only a detailed knowledge of the molecular mechanisms involved will allow us to overcome this barrier.

Commonly invasive serotypes ofStreptococcus pneumoniaetrigger a reduced innate immune response compared with serotypes rarely responsible for invasive infection

Although there are more than 90 serotypes of Streptococcus pneumoniae (or pneumococcus), it is not understood why a small number of serotypes account for most invasive infections. To investigate the human innate immune response triggered by different pneumococcal serotypes, monocyte-derived macrophages were exposed to a group of commonly and rarely invasive pneumococcal clinical isolates and tumor necrosis factor (TNF)-alpha production was measured. Commonly invasive pneumococcal serotypes triggered significantly less TNF-alpha production than serotypes rarely responsible for invasive infection (P<0.004). These data indicate that one factor influencing the invasive potential of a pneumococcal serotype is the magnitude of innate immune-mediated TNF-alpha production triggered by exposure to the organism and suggest that the integrated host response generated against commonly invasive pneumococcal serotypes may be less effective than the response directed against rarely invasive serotypes.

Sphingosine 1-Phosphate 1 and TLR4 Mediate IFN-β Expression in Human Gingival Epithelial Cells

IFN-beta production is a critical step in human innate immune responses and is primarily controlled at the transcription level by highly ordered mechanisms. IFN-beta can be induced by pattern-recognition receptors such as the TLR4. S1P1 is a G protein-coupled receptor, which has a high affinity for sphingosine 1-phosphate (S1P). Although many of the receptors and signaling pathways leading to the expression of IFN-beta have been identified and characterized, it is still unclear how IFN-beta is regulated in primary human gingival epithelial cells (HGECs). In this study, we demonstrate that S1P1 and TLR4, acting in unison, play an important role in IFN-beta expression at the protein and mRNA level in HGECs. We demonstrate that the expression of both IFN-beta and IFN-inducible protein-10 (CXCL-10) is significantly up-regulated by LPS and S1P or LPS and a specific S1P1 agonist. This enhanced innate immune response is attenuated in HGECs by small interfering RNA knockdown of either TLR4 or S1P1. Moreover, we show that triggering of TLR4 results in the increased expression of S1P1 receptors. Furthermore, we found that IFN-regulatory factor 3 activation was maximized by LPS and S1P through PI3K. Our data show that triggering TLR4 increases S1P1, such that both TLR4 and S1P1 acting through PI3K enhancement of IFN-regulatory factor 3 activation increase IFN-beta expression in epithelial cells. The functional association between TLR4 and the S1P1 receptor demonstrates a novel mechanism in the regulation of IFN-beta and CXCL-10 in human primary gingival epithelial cells.

Non-specific Immune Response of Rainbow Trout (Oncorhynchus Mykiss) by Dietary Heat-inactivated Potential Probiotics

Background: The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the rainbow trout were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of rainbow trout, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. Methods: The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10 8 cfu/g Pdp11, 10 8 cfu/g 51M6 or with 0.5×10 8 cfu/g Pdp11 plus 0.5×10 8 cfu/g 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, and then the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. Results: The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in rainbow trout fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. Conclusion: Our results demonstrated the usefulness of incorporating inactivated probiotic bacteria into fish diets. [Immune Network 2008;8(3):67-74]

Substrate and Enzyme Trafficking as a Means of Regulating 1,25-Dihydroxyvitamin D Synthesis and Action: The Human Innate Immune Response

Tissue availability of the active vitamin D metabolite, 1,25-dihydroxyvitamin D [1,25(OH)(2)D] is dependent on expression of the activating enzyme 1alpha-hydroxylase (CYP27b1) and its catabolic counterpart 24-hydroxylase (CYP24). The activity of these two enzymes is in turn controlled by factors including affinity of the serum vitamin D-binding protein (DBP) for 25-hydroxyvitamin D [25(OH)D]; the availability of enzyme cofactors; and the relative amount of hydroxylase gene product expressed. In recent years, it has become clear that directed trafficking of substrate and enzyme is also a pivotal component of the regulated process of hormone synthesis by both renal and extrarenal tissues expressing the CYP27b1 and CYP24 genes. Extracellular regulatory trafficking events are defined by the quantity of substrate 25(OH)D entering the circulatory pool. Entry into some target cells in vivo, such as the macrophage and proximal renal tubular epithelial cells, requires 25(OH)D binding to serum DBP, followed by recognition, internalization, and intracellular release. The "released" intracellular substrate is moved to specific intracellular destinations (i.e., the mitochondrial CYP enzymes and the vitamin D receptor [VDR]) by the hsc70 family of chaperone proteins. Synthesis of 1,25(OH)(2)D is also regulated by CYP24 and its metabolically inactive splice variant CYP24-SV. Finally, initiation of transcription of 1,25(OH)(2)D-regulated genes, such as the CYP24, requires movement of the CYP27b1 product, 1,25(OH)(2)D, to the VDR in the same cell for intracrine action or export to another cell for paracrine action. In either case, the 1,25(OH)(2)D ligand is required for the VDR to heterodimerize with the retinoid x receptor and compete away the dominant-negative acting, heterogeneous nuclear ribonucleoprotein (hnRNP)-related, vitamin D response element-binding proteins that inhibit hormone-directed transactivation of genes. In this review, we use vitamin D-directed events in the human innate immune response to Mycobacterium tuberculosis as a physiologically relevant model system in which to highlight the importance of these intracellular traffic patterns.

Contrasting Infection Strategies in Generalist and Specialist Wasp Parasitoids of Drosophila melanogaster

Although host–parasitoid interactions are becoming well characterized at the organismal and cellular levels, much remains to be understood of the molecular bases for the host immune response and the parasitoids' ability to defeat this immune response. Leptopilina boulardi and L. heterotoma, two closely related, highly infectious natural parasitoids of Drosophila melanogaster, appear to use very different infection strategies at the cellular level. Here, we further characterize cellular level differences in the infection characteristics of these two wasp species using newly derived, virulent inbred strains, and then use whole genome microarrays to compare the transcriptional response of Drosophila to each. While flies attacked by the melanogaster group specialist L. boulardi (strain Lb17) up-regulate numerous genes encoding proteolytic enzymes, components of the Toll and JAK/STAT pathways, and the melanization cascade as part of a combined cellular and humoral innate immune response, flies attacked by the generalist L. heterotoma (strain Lh14) do not appear to initiate an immune transcriptional response at the time points post-infection we assayed, perhaps due to the rapid venom-mediated lysis of host hemocytes (blood cells). Thus, the specialist parasitoid appears to invoke a full-blown immune response in the host, but suppresses and/or evades downstream components of this response. Given that activation of the host immune response likely depletes the energetic resources of the host, the specialist's infection strategy seems relatively disadvantageous. However, we uncover the mechanism for one potentially important fitness tradeoff of the generalist's highly immune suppressive infection strategy.

Transgenic Parasites: Improving Our Understanding of Innate Immunity to Malaria

Clinical immunity to Plasmodium falciparum malaria takes years to develop and is never complete. One explanation for these observations is that antigenic variation enables malaria parasites to evade humoral immunity; another is that P. falciparum induces immune dysregulation, which inhibits the development of protective cellular immunity. Research described by D'Ombrain et al. in this Cell Host & Microbe issue probes how the parasite's main virulence factor PfEMP-1 might significantly alter human innate immune responses.

A κB sequence code for pathway-specific innate immune responses

The Toll and Imd pathways induce humoral innate immune responses in Drosophila by activating NF-kappaB proteins that bind kappaB target sites. Here, we delineate a kappaB site sequence code that directs pathway-specific expression of innate immune loci. Using bioinformatic analysis of expression and sequence data, we identify shared properties of Imd- and Toll-specific response elements. Employing synthetic kappaB sites in luciferase reporter and in vitro binding assays, we demonstrate that the length of the (G)(n) element in the 5' half-site and of the central (A,T)-rich region combine to specify responsiveness to one or both pathways. We also show that multiple sites function to enhance the response to either or both pathways. Together, these studies elucidate the mechanism by which kappaB motifs direct binding by particular Drosophila NF-kappaB family members and thereby induce specialized innate immune repertoires.

Intra- and interspecific relationships between nest size and immunity

Nest-building behavior has been suggested to represent a postmating sexually selected signal in passerine birds, an hypothesis that has received both comparative and experimental support. Because selection pressure due to parasites and diseases should be particularly high during nest building, mainly due to energetic costs and depression of the immune system associated with this reproductive phase, we predicted a positive association between nest-building effort and immunity. Nest-building effort would reflect the ability to produce efficient immune responses of builders only if individuals with a superior immune system would display exaggerated nest-building effort. We tested this prediction by studying the relationship between volume of nest material used for nest construction and, at the intraspecific level, estimates of innate humoral immune response in barn swallows Hirundo rustica. At the interspecific level, we used responses to the mitogenic phytohemagglutinin as an indicator of adaptive immune response of European passerine species. As predicted, we found, after controlling for several potential confounding factors, that volume of nest material was positively related to immune response both at the intra- and at the interspecific level. Alternative hypotheses explaining the comparative results are discussed.

Generation of human innate immune responses towards membrane macrophage colony stimulating factor (mM-CSF) expressing U251 glioma cells within immunodeficient (NIH-nu/beige/xid) mice

The response of human peripheral blood mononuclear cells (PBMC) to cloned human HLA-A2+ U251 glioma cells (U251-2F11/TK) expressing membrane macrophage colony stimulating factor (mM-CSF) was investigated in vitro and in vivo. Enriched human monocytes derived from cancer patients produced a respiratory burst following 20 min of interaction with mM-CSF expressing U251 glioma cells. This respiratory burst response was not observed in the enriched human monocytes following similar exposure to the viral vector control U251 (U251-VV) cells. Reactive oxygen species such as H 2O 2 and HOCl produced death of the U251 cells. The U251-2F11/TK cells failed to grow in severely compromised combined immunodeficient (NIH-bg-nu-xidBR) mice that were depleted of murine monocyte/macrophages then reconstituted with human HLA-A2+ PBMC. Reactive oxygen species (ROS) were produced by PBMC, both in vitro and in vivo in response to mM-CSF expressing U251 cells. U251-2F11/TK cells failed to form subcutaneous tumors in macrophage depleted mice reconstituted with human PBMC; whereas, progressive growth of such tumors was observed with the U251-VV cells. U251-2F11/TK tumors formed if the initial inoculums of PBMC were depleted of monocytes. From this work it can be concluded that mM-CSF transduced U251-2F11/TK glioma cells can safely stimulate human innate immune responses in vivo.

Sharpsnout sea bream ( Diplodus puntazzo) humoral immune response against the parasite Enteromyxum leei (Myxozoa)

The humoral innate immune response of sharpsnout seabream Diplodus puntazzo against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by cohabitation and a group of uninfected fish served as control. At 5, 12, 19, 26, 40 and 55 days post-exposure (p.e.), control and recipient fish were sampled to determine the prevalence of infection and some humoral innate immune parameters (antiprotease, antitumoral and peroxidase activities). Prevalence of infection was high from day 12 p.e. and reached 100% at days 40 and 55, when intensity of infection was medium to severe. The antiprotease activity was significantly increased in E. leei-exposed fish with respect to control fish at days 12 and 19 p.e. The serum antitumoral activity was slightly lower in recipient than in control fish at all sampling times, except at 40 days p.e., though no statistically significant differences were observed. Serum peroxidases were higher in all recipient fish than in control ones, with the highest stimulation index at 40 days p.e. Within recipient fish, no differences were detected between sampling times in any of the measured activities. The possible implication of these immune factors in the high susceptibility of D. puntazzo to this enteromyxosis is discussed.

IRAK-4 Mutation (Q293X): Rapid Detection and Characterization of Defective Post-Transcriptional TLR/IL-1R Responses in Human Myeloid and Non-Myeloid Cells

Innate immunodeficiency has recently been reported as resulting from the Q293X IRAK-4 mutation with consequent defective TLR/IL-1R signaling. In this study we report a method for the rapid allele-specific detection of this mutation and demonstrate both cell type specificity and ligand specificity in defective IL-1R-associated kinase (IRAK)-4-deficient cellular responses, indicating differential roles for this protein in human PBMCs and primary dermal fibroblasts and in LPS, IL-1beta, and TNF-alpha signaling. We demonstrate transcriptional and post-transcriptional defects despite NF-kappaB signaling and intact MyD88-independent signaling and propose that dysfunctional complex 1 (IRAK1/TRAF6/TAK1) signaling, as a consequence of IRAK-4 deficiency, generates specific defects in MAPK activation that could underpin this patient's innate immunodeficiency. These studies demonstrate the importance of studying primary human cells bearing a clinically relevant mutation; they underscore the complexity of innate immune signaling and illuminate novel roles for IRAK-4 and the fundamental importance of accessory proinflammatory signaling to normal human innate immune responses and immunodeficiencies.

Neutrophils from patients with heterozygous germline mutations in the von Hippel Lindau protein (pVHL) display delayed apoptosis and enhanced bacterial phagocytosis

AbstractNeutrophils are key mediators of the innate immune response and are required to function at sites of low oxygenation. We have shown that in hypoxia neutrophils are protected from apoptosis via a mechanism dependent on prolyl hydroxylase domain/hypoxia-inducible factor 1α (PHD/HIF-1α). This response would be predicted to involve the von Hippel Lindau protein (pVHL)–dependent ubiquitination and degradation of HIF-1α. Patients with VHL disease inherit a mutation in one VHL allele, which allows us to study the effects of heterozygous VHL expression in human neutrophils. Neutrophils exhibited a striking “partial hypoxic” pheno-type, with delayed rates of apoptosis and enhanced bacterial phagocytosis under normoxic conditions and preserved responses to low levels of oxygen. This provides direct evidence that the HIF-1α/VHL pathway regulates the innate immune response in humans. It also establishes that heterozygous VHL defects are sufficient to perturb normal responses and illustrates the potential to use this to address the role of HIF and VHL in human biology.

Effect of photoperiod on the fish innate immune system: a link between fish pineal gland and the immune system

The pineal gland via its secretory product, melatonin, influences the light-dark rhythm in most vertebrates including fish. Apart from the information concerning this circadian rhythm, the interrelation of the melatonin with other physiological processes has not been considered in fish. Thus, we evaluated the changes in the humoral innate immune system of seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) specimens exposed to a constant light-dark photoperiod (12 hr L:12 hr D). Serum was obtained from blood samples collected at 02:00, 08:00 hr (light-on), 14:00, 20:00 hr (light-off) and at 08:00 hr again. Among the humoral innate immune responses, complement, lysozyme and peroxidase activities were determined. Complement activity was higher during the day than during the night in both fish species. Seabream lysozyme activity reached its maximum at 20:00 and 02:00 hr but was hardly affected in sea bass. Finally, the peroxidase activity of seabream was significantly higher at 08:00 hr than during the rest of the cycle while, in sea bass, it showed little variation. The present results demonstrate that the humoral innate immune system has a circadian rhythm based on the light-dark cycle and that this cycle might be affected by the pineal gland.

EFFECT OF SELECTION FOR HIGH AND LOW ANTIBODY RESPONSE AGAINST SHEEP RED BLOOD CELLS ON HORMONAL PROFILE DURING IMMUNE RESPONSE IN CHICKENS

SUMMARY Two experiments were conducted in this study using two lines of Fayoumi chickens divergently selected for high (H) and low (L) antibody response against sheep red blood cells (SRBC) for four generations. The objectives of this study are to compare humoral and cell mediated immune responses between the lines, as well as to monitor the T3 and corticosterone hormone profile during the initiation of the immune responses. In the first experiment one hundred male chicks of each line were primary injected intramuscular with either 1 ml 25% SRBC suspension or saline at 50 and secondary reinjected at 80 days of age. Blood samples were collected at 0, 1, 3, 6, 9, 12 and 24 h and at 3, 7 and 10 days following primary and secondary injection. The results showed that H line produced significantly higher primary and secondary antibody titer than L line at 7 and 10 days post injection. The results showed that T3 levels decreased significantly at 3 h in H line and at 3, 6 and 9h in L line, then remained at low levels till 10 days post primary SRBC injection as compared to 0 h. Within both H and L lines, the T3 level showed insignificant differences between 0 hr and at any time post secondary SRBC or saline injection ( except that in H line at 3h post SRBC injection). In both line there was a significant increase in corticosterone levels at 24h as well as at 3, 7 and 10 days post primary, and at 3,7 and 10 days post secondary SRBC injection as compared to 0 h. In the second experiment, one-hundred and twenty male chicks of each line were injected intradermally into wattle with either 100 μg PHA-P or saline at 56 days of age to monitor the delayed type hypersensitivity (DTH) reaction. Blood samples were collected at 0, 3, 6, 9, 12 and, 24 hs post injection. The results indicate that H line had higher DTH value at 12 and 24 h than that in L line at the same time point. There was no remarked change in the level of T3 or corticosterone between PHA-P and saline injected birds at any time point following injection within both lines. In conclusion, the results of T3 or corticosterone levels in the current study may explain that the initiation of humoral immune response involves the activation of HPT- and HPA-axis. While this axis is not involved in delayed type hypersensitivity reaction.

Dominance of human innate immune responses in primary Francisella tularensis live vaccine strain vaccination

Abstract : Francisella tularensis is the etiologic agent of the zoonotic disease, tularemia. An inoculum as small as 10 bacteria can cause a flulike disease with substantial morbidity and mortality among infected humans. Human tularemia presents in ulceroglandular, oculoglandular, oropharyngeal, pneumonic, and septic forms. Rapid administration of antibiotics prevents mortality in the majority of human cases if exposure doses are low and nonaerosol. Without early diagnosis and administration of antibiotics, high-dose aerosol exposure progresses rapidly to life threatening pleuropneumonitis and systemic infection. The relative abundance of F tularensis in nature and the relative ease with which it may be administered raise concerns over its exploitation as a biothreat agent.

Cell-Cell Interactions and Cell Fate Determination: Involvement of Dlk in B Cell Developmentand Function

RATIONALE: Dlk (Delta like-1) is expressed on a subset of stroma and regulates HSC proliferation. We created a dlk -/- mouse model to investigate the importance of the stromal microenvironment for proper development and function of B cells. METHODS: FACS analysis of B cell subsets; immuno-histochemical analysis of spleen sections; in vitro differentiation of bone marrow B cells; ELISA on serum Ig before and after DNP-KLH challenge. RESULTS: Compared to wild type controls, bone marrow B cells from dlk -/- mice had a 30% increase in fraction-A cells (4-week-old mice, n=4,P<0.01), and a 2.4-fold decrease in Fraction-C cells. (P< 0.0001, 6-week old mice, n=4). In vitro differentiation of total bone marrow cells supplemented with IL7 resulted in increased (4-fold) differentiation of fraction C. Mature recirculating B cell numbers decreased 2-3 fold. Spleens of 6-week-old mice had two-fold increases in Tr1 cells. Eight-week-old mice had increased marginal zone B cells and decreased follicular B cells (P < 0.001, n=3). Serum IgG was high and upon immunization it increased dramatically for DNP specific responses (n=4 P<0.01). CONCLUSIONS: The alterations in the stroma dependent progenitor B cells, the splenic B cell subsets and the hyper IgG response indicate dlk is important in development and function of normal B cell. These results suggest that dlk could be a useful target for modulating innate humoral immune responses.

Prevalence and Pathology of Amyloid in Atherosclerotic Arteries

To the Editor: Apolipoprotein AI (AApoAI)–associated amyloidosis is characterized by the deposition of apolipoprotein AI (apoAI) and occurs as a hereditary and a nonhereditary form. Hereditary AApoAI amyloidosis is a systemic disease leading to the deposition of amyloid in various organs and tissues and is caused by germline mutations in the APOA1 gene. Nonhereditary AApoAI amyloid is far more prevalent and characterized by deposits of nonvariant protein in atherosclerotic arteries.1–3 Despite being linked to the most common cause of morbidity and mortality in Western societies, nonhereditary AApoAI amyloid has achieved only little attention.1–4 It shares several striking similarities with secondary or reactive AA amyloidosis. Nonhereditary AApoAI amyloid occurs in the background of a local chronic inflammatory reaction, it originates from an apolipoprotein that largely associates with high-density lipoproteins (HDLs), and apoAI-containing HDL is endocytosed and retroendocytosed by macrophages, which, in themselves, are able to form amyloid in vitro. Finally, AApoAI amyloidosis is characterized by the deposition of proteolytic fragments of the precursor protein, leading us to speculate that proteolysis is involved in the pathogenesis of AApoAI amyloid.1,3 The aim of this study was to gain further insights into the pathology of nonhereditary AApoAI amyloid. See cover The prevalence and spatial distribution of amyloid, macrophages, cathepsin B (CathB), cathepsin K (CathK), cathepsin L (CathL), and carboxy methyl lysine (CML) was studied using carotid artery specimens obtained from a consecutive series of all 225 patients undergoing carotid endarterectomy with polyester patch angioplasty (Table I, available online at http://atvb.ahajournals.org) during the period from 1997 to 2003. All patients were scheduled for elective therapeutic endarterectomy and gave written informed consent in the surgical procedure. Patient characteristics were retrieved from hospital records. This study was in accordance with the guidelines of the ethics committee of the University of Magdeburg. Tissue samples were …

Intestinal Innate Immunity toCampylobacter jejuniResults in Induction of Bactericidal Human Beta-Defensins 2 and 3

Campylobacter jejuni is the most prevalent cause of bacterial diarrhea worldwide. Despite the serious health problems caused by this bacterium, human innate immune responses to C. jejuni infection remain poorly defined. Human beta-defensins, a family of epithelial antimicrobial peptides, are a major component of host innate defense at the gastrointestinal mucosal surface. In this study, the effect of two different C. jejuni wild-type strains on human intestinal epithelial innate responses was investigated. Up-regulation of beta-defensin gene and peptide expression during infection was observed and recombinant beta-defensins were shown to have a direct bactericidal effect against C. jejuni through disruption of cell wall integrity. Further studies using an isogenic capsule-deficient mutant showed that, surprisingly, the absence of the bacterial polysaccharide capsule did not change the innate immune responses induced by C. jejuni or the ability of C. jejuni to survive exposure to recombinant beta-defensins. This study suggests a major role for this family of antimicrobial peptides in the innate immune defense against this human pathogen.

Gilthead seabream ( Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10 8 cfu g −1 Pdp11, 10 8 cfu g −1 51M6 or with 0.5 × 10 8 cfu g −1 Pdp11 plus 0.5 × 10 8 cfu g −1 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.