Innate immunity and vaccination.
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Animals↗
Biomedical subjects
Pete Kaiser
Publications and source records attributed to Pete Kaiser.
Re-evaluation of the chicken MIP family of chemokines and their receptors suggests that CCL5 is the prototypic MIP family chemokine, and that different species have developed different repertoires of both the CC chemokines and their receptors.
Analysis of the chicken genome has shown that the chicken has a different repertoire of chemokines and chemokine receptors to those of mammals and other species. In this study, we report the sequencing and analysis of a bacterial artificial chromosome containing the entire chicken MIP family CC chemokine cluster. The gene duplication and divergence events that have taken place in mammals do not appear to have occurred as extensively in the avian lineage, as chickens possess fewer MIP family chemokine genes than humans or mice. We previously proposed that the four chicken MIP family members be named chicken (ch) CCLi1-4, according to their position on chicken chromosome 19, until such time as further analysis could determine if any of them were direct orthologues of mammalian MIP family members. Our analysis herein, combined with that of others, suggests that chCCLi4 is the orthologue of mammalian CCL5, and that chCCLi3 (K203) may be an orthologue of human CCL16. The other two chemokines do not have obvious orthologues, and thus we propose that they should still be called chCCLi1 and chCCLi2, until their biological function is further characterised. A similar pattern applies to the MIP family chemokine receptors, with only three receptor genes present at the relevant locus in the chicken genome, compared to four in man and mouse (CCR1, CCR2, CCR3 and CCR5). Of the three chicken receptor genes, only two look likely to be receptors for the MIP family chemokines, the third grouping with human, mouse and chicken CCR8 in phylogenetic analysis. The two chicken MIP CC receptors (CCRs) are not direct orthologues of the mammalian MIP CCRs.
Amino Acid Sequence↗
Breed effect on early cytokine mRNA expression in spleen and cecum of chickens with and without Salmonella enteritidis infection.
We examined mRNA expression of 11 genes: BAK, Bcl-x, Interferon [IFN]-gamma, Interleukin [IL]-1beta, IL-6, IL-10, IL-12alpha, IL-12beta, IL-18, CXCLi2 [IL-8/CAF], and a MIP family chemokine, CCLi2, in the spleen and cecum of day-old chicks after oral inoculation with Salmonella enteritidis (SE) or medium. Three distinct chicken breeds (broiler, Fayoumi, and Leghorn) were evaluated for mRNA expression levels at 2 and 18h post-inoculation using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). SE exposure significantly increased splenic IL-18 and IFN-gamma expression. Breed effect was significant (P<0.05) for CXCLi2, IL-10, IL-12alpha, and CCLi2 mRNA expression in the spleen, and for IL-12alpha, IL-12beta, IL-18, and CCLi2 mRNA expression in the cecum. Generally, mRNA expression levels were higher in the spleen, and lower in the cecum, of Leghorns versus broilers. These results support a role for breed genetics influencing cytokine mRNA expression in young chickens and may potentially explain some generalized immune response differences between breeds.
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Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses.
Heterophils isolated from distinct broilers (lines A and B) differ in function and cytokine gene expression profiles. Nothing is known about Toll-like receptor (TLR) expression nor functional activation and cytokine/chemokine gene expression of line A and B heterophils when stimulated with TLR agonists. We found that line A and B heterophils express the same range of TLRs. All the bacterial TLR agonists, peptidoglycan, the synthetic lipoprotein Pam3CSK4, ultra-pure lipopolysaccharide, and flagellin all induced significantly greater functional activation of heterophils from line A compared to B. Only stimulation with the guanosine analog, loxoribine, (LOX) induced a significantly greater functional response in B over A. Additionally, all heterophils from line A stimulated with the bacterial TLR agonists had dramatic upregulation of pro-inflammatory cytokine and chemokine mRNA expression, whereas heterophils from line B had little or no upregulation of these genes. However, stimulation of all heterophils from line B with the bacterial TLR agonists and LOX induced a significant upregulation of IFN-alpha, with little transcription of this cytokine gene in line A heterophils. These findings suggest that the difference in heterophil functional efficiency between these parent lines is due to recognition of pathogens and activation of signaling pathways that induce innate cytokine and chemokine responses.
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Heterophil cytokine mRNA profiles from genetically distinct lines of chickens with differential heterophil-mediated innate immune responses.
Previously we demonstrated that increased in-vitro heterophil function translates to increased in-vivo resistance to Salmonella enteritidis infections in broilers (line A > B). Heterophils produce cytokines and modulate acute protection against Salmonella in neonatal poultry. We hypothesized that heterophils from S. enteritidis-resistant chickens produce an up-regulated pro-inflammatory cytokine/chemokine response compared with S. enteritidis-susceptible chickens. In this study, heterophils were isolated 1, 14, and 28 days post-hatch, treated with RPMI or phagocytic agonists, and the cytokine/chemokine mRNA expression assessed using quantitative real-time reverse transcriptase-polymerase chain reaction. At all time-points, heterophils from S. enteritidis-resistant chickens (line A) had higher levels of pro-inflammatory cytokine/chemokine mRNA expression upon stimulation compared with heterophils from S. enteritidis-susceptible chickens (line B). Furthermore, heterophils from line A chickens had decreased mRNA expression of transforming growth factor-beta4, an anti-inflammatory cytokine, compared with line B. These data indicate a relationship between cytokine/chemokine mRNA expression by heterophils and determining overall immune competence. Therefore, heterophil functional efficiency, accompanied by evaluating cytokine/chemokines produced by heterophils, may be useful biomarkers for breeders to consider when developing new immunocompetent lines.
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The feathering gene is linked to degranulation and oxidative burst not cytokine/chemokine mRNA expression levels or Salmonella enteritidis organ invasion in broilers.
In the past, we showed differences in heterophil function between parental broilers (A [fast feathering] > B [slow feathering]) and their F1 reciprocal crosses (D [fast feathering] > C [slow feathering]). In the present study, we evaluated the linkage of the feathering gene to heterophil function, pro-inflammatory cytokine/chemokine mRNA expression levels, and resistance to Salmonella enteritidis organ invasion. Heterophils were isolated from 2-day-old chickens (C and D) separated into males and females - slow males and females (SM and SF), and fast males and females (FM and FF). Heterophil functions of degranulation and oxidative burst were measured. Heterophils from FF chickens (183+/-8.9) released more (P < 0.05) beta-d-glucuronidase (microM) than heterophils from SF chickens (149+/-3.7); FF heterophils (4.6 x 10(4)) generated a significantly (P < 0.05) greater oxidative burst (mean relative fluorescent units) compared with SF heterophils (4.2 x 10(4)). Interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were quantitated by real-time quantitative reverse transcriptase-polymerase chain reaction. No differences were observed between SM and FM or between SF and FF heterophils. Finally, 1-day-old chickens were administered S. enteritidis and liver/spleen organ invasion was quantitated. No differences were observed between the number of S. enteritidis-positive FF and SF chickens, but FM were significantly (P < 0.05) more resistant to S. enteritidis organ invasion than SM chickens. The data indicate degranulation and oxidative burst were linked with the feathering gene; however, interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were not. Furthermore, susceptibility to in vitro S. enteritidis organ invasion was not linked to the feathering gene.
Aging↗
Infectious bursal disease virus: strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa.
Little is understood about the immune responses involved in the pathogenesis of infectious bursal disease virus (IBDV). Strains of IBDV differ in their virulence: F52/70 is a classical virulent strain (vIBDV), whereas UK661 is a very virulent strain (vvIBDV) that causes greater pathology and earlier mortality. The exact causes of clinical disease and death are still unclear. Pro-inflammatory cytokines such as interleukin (IL)-1beta and IL-6, produced by activated macrophages, could play a role, as could cytokines produced by T and natural killer (NK) cells, such as interferon (IFN)-gamma, which stimulate macrophages. We quantified mRNA transcription in bursal tissue, by real-time quantitative reverse transcription- polymerase chain reaction (RT-PCR), for the type I IFN (IFN-alpha and IFN-beta), pro-inflammatory cytokines (IL-1beta, IL-6, and CXCLi2), the anti-inflammatory cytokine transforming growth factor (TGF)-beta4, and Th1 cytokines (IFN-gamma, IL-2 [and the closely related IL-15], IL-12, and IL-18) for the first 5 days after infection of 3-week-old chickens with F52/70 or UK661 and compared these with levels in bursal tissue from uninfected age-matched controls. Both strains induced a pro-inflammatory response, evidenced by increased mRNA transcription of IL-1beta, IL-6, and CXCLi2, and down-regulation of TGF-beta4, of similar magnitude and timing. IFN-gamma mRNA was induced by both strains, although to a greater degree by the vvIBDV strain, indicating that a cell-mediated response is induced. Neither virus initially induced high levels of type I IFN. F52/70 seems to use a "stealth" approach by not inducing the type I IFNs, whereas UK661 down-regulates their expression. This suggests that both viruses modulate the host immune response, although probably by using different mechanisms.
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Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge.
Macrophages from inbred chickens that are resistant to salmonellosis show greater and more rapid expression of proinflammatory chemokines and cytokines, including the key Th1-inducing cytokine interleukin-18, upon Salmonella challenge than those from susceptible birds. This suggests the possibility that salmonellosis resistant-line macrophages signal more effectively and rapidly and are more able to induce protective Th1 adaptive responses.
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Nucleolin is regulated both at the level of transcription and translation.
Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally.
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Expression and function of Toll-like receptors in chicken heterophils.
The heterophil is the major polymorphonuclear cell in birds with a functional capacity akin to that of the mammalian neutrophil. Herein, we demonstrate that heterophils constitutively express TLR1/6/10, TLR2 type 1, TLR2 type 2, TLR3, TLR4, TLR5, and TLR7 mRNA. Furthermore, TLR agonists, including flagellin (from Salmonella typhimurium, FGN), peptidoglycan (from Staphylococcus aureus, PGN), ultra-pure lipopolysaccharide (from Salmonella minnesota, LPS), the synthetic double stranded RNA analog [poly(I:C)], and the guanosine analog, loxoribine (LOX) directly induced both an oxidative burst and a degranulation response. Interestingly, the synthetic bacterial lipoprotein Pam3CSK4 (palmitoyl-3-cysteine-serine-lysine-4, PAM) induced degranulation, but no oxidative burst. The bacterial TLR agonists (PAM, PGN, LPS, and FGN) all induced an up-regulation of expression of mRNA of the pro-inflammatory cytokines IL-1beta, IL-6, and IL-8; whereas both poly(I:C) and LOX induced a down-regulation of these cytokine mRNAs. Stimulation of heterophils with each specific TLR agonist led to a differential increase in the phosphorylation of both p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation, but not the phosphorylation of c-Jun NH2-terminal kinase (JNK). The broad TLR expression profile in heterophils reflects their principal role as first line effector cells in avian host defense against bacterial, viral, fungal, and parasitic infections. The results demonstrate the differential involvement of TLR-induced signals in the stimulation of transduction pathways that regulate the oxygen-dependent and -independent antimicrobial defense mechanisms of avian heterophils.
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Th1/Th2 polarization by viral and helminth infection in birds.
Mammals developed an immune system able to functionally polarize into so-called type 1 or type 2 immune pathways, to resolve infections with intracellular and extracellular pathogens, respectively. In the well-studied avian immune system of the chicken, however, no evidence for polarized immunity could be found, as yet. To investigate whether these two major arms of mammalian immunity, regulated by a T helper (Th)1/Th2 cytokine balance, evolved similarly in birds, chickens were exposed to a prevalent intracellular (viral) or extracellular (helminth) infection. By using semi-quantitative RT-PCR analysis we provide evidence that polarization of Th1/Th2 type immunity extends beyond mammalian species, and, therefore, has been evolutionary conserved for more than 300 million years, when the lineages of mammalian and avian vertebrates are assumed to have segregated.
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Recombinant chicken IL-6 does not activate heterophils isolated from day-old chickens in vitro.
Pro-inflammatory cytokines are produced as part of innate immunity. Increased resistance to extraintestinal Salmonella enteritidis (SE) has been associated with an increase in heterophil pro-inflammatory cytokine gene expression. Invasion of chicken epithelial cells by SE induces an 8- to 10-fold increase in interleukin (IL) -6 production. Infection with SE induces an influx of heterophils to the site of infection; therefore, we hypothesize heterophils would be responsive to IL-6. The objective was to determine the effects of COS cell-derived recombinant chicken interleukin 6 (rChIL-6) on in vitro functional activity of heterophils. Heterophils were incubated with rChIL-6 or mock-transfected COS cell supernatant and functional activity was assessed. Heterophils treated with rChIL-6 showed no functional differences compared to controls. These data indicate rChIL-6, alone, does not affect the functional activity of neonatal chicken heterophils in vitro. Therefore, the function of IL-6 in the local environment in response to SE invasion is still unknown.
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Lipopolysaccharide binding protein/CD14/ TLR4-dependent recognition of salmonella LPS induces the functional activation of chicken heterophils and up-regulation of pro-inflammatory cytokine and chemokine gene expression in these cells.
Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern (PAMP) found in the cell wall of gram-negative bacteria and, in mammals, is recognized by the Toll-like receptor 4 (TLR4) in conjunction with the serum protein, lipopolysaccharide-binding protein (LBP), and the CD14 co-receptor. We have found that chicken heterophils constitutively express multiple TLRs including TLR4. Interestingly, ultrapure LPS from Salmonella minnesota directly induced the functional activation of heterophils without the presence of LBP. However, the role of LBP and CD14 in the recognition of LPS and the induction of innate immunity, including cellfunctional activation and the transcription of cytokine and chemokine genes in chicken heterophils, is not known. As previously seen, in the absence of chicken serum, heterophil exposure to ultrapure LPS from Salmonella minnesota stimulated an increased degranulation response. However, the presence of 5% chicken serum, presumed to be a source of LBP, increased heterophil degranulation by 84%. In addition, the presence of either soluble recombinant human LBP (rhLBP, 68%) or CD14 (39%) also induced the up-regulation of the heterophil degranulation response. Incubation of heterophils with either chicken serum or rhLBP also significantly induced the up-regulation of pro-inflammatory cytokine (IL-1beta, IL-6, and IL-18) and chemokine (CCLi4, CXCLi1, CXCLi2, and the CXC receptor 1) mRNA expression. Moreover, polyclonal antibodies directed against rat CD14 and human TLR4, but not antibodies against human TLR2, blocked LPS-mediated degranulation and up-regulation of the pro-inflammatory cytokine and chemokine mRNA expression. These data clearly demonstrate that LBP and CD14/TLR4 engagement is directly involved in LPS-mediated functional activation and innate immune gene expression in chicken heterophils.
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A genomic analysis of chicken cytokines and chemokines.
As most mechanisms of adaptive immunity evolved during the divergence of vertebrates, the immune systems of extant vertebrates represent different successful variations on the themes initiated in their earliest common ancestors. The genes involved in elaborating these mechanisms have been subject to exceptional selective pressures in an arms race with highly adaptable pathogens, resulting in highly divergent sequences of orthologous genes and the gain and loss of members of gene families as different species find different solutions to the challenge of infection. Consequently, it has been difficult to transfer to the chicken detailed knowledge of the molecular mechanisms of the mammalian immune system and, thus, to enhance the already significant contribution of chickens toward understanding the evolution of immunity. The availability of the chicken genome sequence provides the opportunity to resolve outstanding questions concerning which molecular components of the immune system are shared between mammals and birds and which represent their unique evolutionary solutions. We have integrated genome data with existing knowledge to make a new comparative census of members of cytokine and chemokine gene families, distinguishing the core set of molecules likely to be common to all higher vertebrates from those particular to these 300 million-year-old lineages. Some differences can be explained by the different architectures of the mammalian and avian immune systems. Chickens lack lymph nodes and also the genes for the lymphotoxins and lymphotoxin receptors. The lack of functional eosinophils correlates with the absence of the eotaxin genes and our previously reported observation that interleukin- 5 (IL-5) is a pseudogene. To summarize, in the chicken genome, we can identify the genes for 23 ILs, 8 type I interferons (IFNs), IFN-gamma, 1 colony-stimulating factor (GM-CSF), 2 of the 3 known transforming growth factors (TGFs), 24 chemokines (1 XCL, 14 CCL, 8 CXCL, and 1 CX3CL), and 10 tumor necrosis factor superfamily (TNFSF) members. Receptor genes present in the genome suggest the likely presence of 2 other ILs, 1 other CSF, and 2 other TNFSF members.
Amino Acid Sequence↗
IFN-gamma priming of chicken heterophils upregulates the expression of proinflammatory and Th1 cytokine mRNA following receptor-mediated phagocytosis of Salmonella enterica serovar enteritidis.
Responsiveness to invasive pathogens, clearance via the inflammatory response, and activation of appropriate acquired responses are all coordinated by innate host defenses. Polymorphonuclear leukocytes (PMNs) are cellular components of innate response, with the primary PMN in poultry being the heterophil. Priming is the potentiation of the phagocyte activation process. Interferon-gamma (IFN-gamma) is a pleiotropic cytokine involved in basically all phases of immune and inflammatory responses that has been shown to prime heterophil functional activities. In the present experiments, using real-time quantitative RT-PCR, we evaluated the role of recombinant chicken IFN-gamma (rChIFN-gamma) as a priming mediator to control heterophil responses at the level of gene transcription and expression of the mRNA for proinflammatory (interleukin-1beta [IL-1beta], IL-6, IL-8) and Th1 (IL-18 and IFN-gamma) cytokine genes following stimulation with phagocytosis agonists, opsonized and nonopsonized Salmonella enteritidis. rChIFN-gamma primed the heterophils for an increase in transcription of proinflammatory cytokines induced by phagocytic agonists but also upregulated expression of Th1 cytokine (IL-18 and IFN-gamma) mRNA and stimulated an increased production of IFN-gamma. Although rChIFN-gamma priming modulated the expression of cytokine mRNA in heterophils stimulated by different phagocytic agonists, rChIFN-gamma by itself did not directly induce gene expression of either proinflammatory or Th1 cytokines. The enhanced expression of cytokine mRNA does not appear to be differentially expressed depending on the receptor activated during phagocytosis. The results from the present experiments suggest that rChIFN-gamma may play a significant role in avian innate immunity against Salmonella infection and may offer an adjunct use in the prevention and treatment of salmonellae infections in newly hatched chickens.
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A strong antigen-specific T-cell response is associated with age and genetically dependent resistance to avian enteric salmonellosis.
Chicken genetics and age affect resistance to enteric infection with Salmonella enterica serovar Typhimurium and were used to identify the immune responses that may contribute to rapid clearance. When birds were infected at 40 days of age, line 6(1) chickens cleared the infection more effectively than line N chickens, whereas when birds were infected at 10 days of age, both chicken lines were highly susceptible to infection. Antibody levels, T-cell responsiveness, and cytokine mRNA levels were all elevated during infection. A negative correlation between resistance and antigen-specific antibody production was observed in older chickens. However, this finding was not replicated for age-related resistance; we found that older chickens exhibited a stronger and more rapid antibody response than younger chickens. The levels of interleukin-1beta (IL-1beta) and gamma interferon (IFN-gamma) mRNA were similar in the spleens and cecal tonsils of both line 6(1) and line N chickens, except for higher levels of IL-1beta in the spleens of line 6(1) chickens at 6 days postinfection. Differences in the levels of IFN-gamma and IL-1beta 1beta mRNA between the lines were more apparent in younger chickens, but while the increases were greater than those observed in the older chickens, the clearance of enteric S. enterica serovar Typhimurium was much slower. The level of antigen-specific proliferation of splenocytes was associated with increased resistance in both experimental systems, and the strongest responses were observed in older and genetically resistant chickens. The data presented here implicate T-cell responses in the clearance of S. enterica serovar Typhimurium from the intestine of infected chickens.
Aging↗
Sublethal infection of C57BL/6 mice with Salmonella enterica Serovar Typhimurium leads to an increase in levels of Toll-like receptor 1 (TLR1), TLR2, and TLR9 mRNA as well as a decrease in levels of TLR6 mRNA in infected organs.
Sublethal infection of C57BL/6 mice with Salmonella enterica serovar Typhimurium M525P initiates a strong inflammatory response. We measured organ expression of mRNA for Toll-like receptors and their associated signaling molecules during S. enterica serovar Typhimurium infection. During infection, the Toll-lie receptor 1 (TLR1), TLR2, and TLR9 mRNA levels increased, while TLR6 mRNA expression decreased.
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Campylobacter jejuni-induced cytokine responses in avian cells.
Campylobacter jejuni is a major cause of human inflammatory enteritis. During the course of human disease numerous proinflammatory cytokines are produced. Little is known, however, about the cytokine responses produced during the interaction of this bacterium with the avian host. Campylobacter has been considered a commensal of the avian host. Any differences in innate responses to this pathogen between the human and avian hosts should lead to a greater understanding of the disease process in humans. We have demonstrated expression of proinflammatory cytokines and chemokines in response to Campylobacter infection in avian primary chick kidney cells and the avian macrophage cell line HD11. The data indicate that Campylobacter can stimulate the avian host in a proinflammatory manner. The data strongly suggest that the lack of pathology in vivo is not due to an inability of Campylobacter to stimulate a proinflammatory response from avian cells.
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