Infection and Immunity/Mucosal Immunology Workshop
Sunday 11th December 2011
Venue: Riverbank Room 2, Adelaide Convention Centre
Session I: Innate and Adaptive Immunity
8:30 AM - 10:15 AM
Chair: Erin Lousberg and Andrew Currie
8:30am
Erin Lousberg - Welcome
8:35am
Paul Kubes (University of Calgary)
“Neutrophil responses in sterile and infectious inflammation”
9:10am
Andrew Currie (Murdoch University)
“Innate immunity in the newborn: to in utero and beyond!”
9:40am
Christina Ziegler (Helmholtz Centre for Infection Research)
“The dynamics of T cells during persistent Staphylococcus aureus infection: from antigenreactivity to in vivo anergy”
9.55 am
Danushka Wijesundara (John Curtin School of Medical Research)
“Interleukin (IL)-4/ IL-13 receptor distribution on immune cells following vaccinia virus infection:
implications on CD8+ T cell functionality”
Morning Tea
10:15 AM - 10:45
Session II: Immune response to bacterial pathogens
10:45 AM - 12:35 PM
Chair: Gabriela Minigo and Ken Beagley
10:45am
Deborah Strickland (TICHR and Centre for Child Health Research, UWA)
“Potential novel therapeutic strategies for control of allergic airways disease”
11:15am
Karren Plain (University of Sydney)
“Role of IDO and tryptophan catabolism in chronic mycobacterial disease pathogenesis”
11:35am
Kumi de Silva (University of Sydney)
“Immune correlates of protection and susceptibility to mycobacterial disease”
11:50am
Natkunam Ketheesan (James Cook University)
“The deadly duo: how a tropical infection decimates a diabetic host”
12:05pm
Andrew Mitchell (University of Sydney)
“NK cell-derived IFNγ contributes to pathology in a murine model of Streptococcus pneumonia
meningitis”
12:20pm
Natalie Lorenz (University of Auckland)
“SSL7 – S. aureus’s answer to the complement system”
Lunch
12:35 PM - 1:30 PM
Session III: Inflammation and Disease
1:30 PM - 2:35 PM
Chair: Matt Sweet and Nick Gorgani
1:30pm
Richard Flavell (Yale University)
“Tolerance, Tissue Defence and the Th17 response”
2:05pm
Seth Masters (WEHI)
“Activation of the Nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for
enhanced IL-1Beta in type 2 diabetes”
Afternoon Tea
2:35 PM – 2:50 PM
Session IV: Mucosal Immunology
2:50 PM - 4:30 PM
Chair: Phil Sutton and Odilia Wijburg
2.50pm
Astrid Westendorf (University of Duisburg-Essen)
“Generation and function of immunosuppressive CD4+ and CD8+ Foxp3+ regulatory T cells in
the intestinal mucosa”
3:25pm
Marina Harvie (Queensland University of Technology)
“Lung localised CD4 T cells are sufficient for protective immunity against helminth infection in
the absence of T cell recirculation”
3:40pm
Margaret Dunkley (Hunter Immunology Ltd)
“Protection against respiratory bacterial infection by oral immunisation”
3:55pm
Alison Hodgkinson (AgResearch)
“Effects of milk and soy protein-based diets on caecal microbiota profiles in the Interleukin-10
gene-deficient mouse”
4:10pm
Yang Xi (John Curtin School of Medical Research)
“Role of Novel type I Interferon Epsilon (IFN-ε) in viral infection and mucosal immunity”
4:30pm
Close of workshop
S e s s i o n I : In n a t e a n d A d a p t i v e Im m u n i t y
8.30 AM – 10.15 AM
“Neutrophil Responses in Sterile and Infectious Inflammation”
P. Kubes
University of Calgary
It is well known that in most vascular beds neutrophils and other cell types are recruited primarily in the post-capillary
venules making use of endothelial selectins including P-selectin and E-selectin to initially tether and roll. Integrins
were then absolutely required for subsequent adhesion and crawling to sites of emigration into the surrounding
tissue. By contrast, close to 90% of neutrophils during infections adhere in the capillaries of sinusoids of the liver and
this occurs independent of selectins or integrins. In fact, a key molecule in infectious immunity for neutrophil
recruitment appeared to be CD44 and its associated molecule SHAP. The neutrophils primarily adhered and did not
crawl. However, following sterile inflammation, the recruitment was strikingly different in liver. The neutrophils
adhered primarily in the sinusoids but immediately began crawling in the vasculature towards the injury. The
recruitment was not dependent at all on CD44 and required the integrin CD11b/CD18 and ICAM-1. Chemokines
deposited on the endothelial surface were key to this recruitment as were mitochondrial derived formylated peptides.
We were intrigued to find that during infectious responses, platelets bound avidly to the neutrophils and induced the
release of Neutrophil Extracellular Traps or NETs. These were critical to catching bacteria in the vasculature and
dramatically increased the liver's bacterial filtering capacity. NETs could also be seen being formed in response to
S.aureus outside the vasculature. In this particular case there was strong evidence for a non-lytic form of NET
formation where the neutrophils continued to function in the tissue.
“Innate immunity in the newborn: to in utero and beyond”
A. Currie1,2, T. Strunk2,3, J. Hibbert2, A. Prosser2, K. Simmer2,3,4, P. Richmond2, D. Burgner2,7
1School
of Veterinary & Biomedical Sciences, Murdoch University, Perth, Western Australia,
School of Paediatrics and Child Health, University of Western Australia, Perth, Western
3Department of Neonatal Paediatrics, King Edward Memorial and Princess Margaret Hospitals, Australia,
4School of Women’s and Infants’ Health, University of Western Australia, Perth, Western Australia,
5Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Australia
2
Infections with extracellular bacteria such as Streptococci and Staphylococci, account for considerable mortality and
morbidity in early childhood, especially in those born prematurely. Susceptibility is largely dependent on innate
immune development, but the innate defence pathways responsible for recognition and control of such Gram-positive
bacteria are poorly characterised in the newborn. Moreover it is unknown: (i) whether these defence pathways differ
in those at higher risk of infection (such as extremely preterm infants or those in developing countries); (ii) if these
differences persist beyond infancy and; (iii) how these innate immune pathways are regulated in utero and from birth.
We have been addressing these questions using a range of prospective, longitudinal and cross-sectional cohorts of
cord and peripheral blood samples collected from preterm and term infants. Using preterm infants of different
gestational ages, with differing degrees of susceptibility to infection, we have been able to map out the ‘in utero’
development of monocyte phagocytic, antimicrobial, and pattern recognition receptor (TLR, NLR and other cytsolic
pathways) function. We find that while many innate defences appear to develop early in gestation, TLR-induced
responses and the ability to respond to cytosolic dsDNA appear to lag behind. We are currently exploring the
epigenetic regulation and postnatal maturation of these pathways, and the impact of prenatal exposure to factors
such as intrauterine infection and inflammation on their development.
“The dynamic of T cells during persistent Staphylococcus aureus infection: from antigenreactivity to in vivo anergy”
C. Ziegler1, O. Goldmann1, E. Hobeika2, R. Geffers3, G. Peters4, E. Medina1
1
Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Immunology, Max Planck Institute for Immunobiology, Freiburg, Germany
3Cell Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
4Medical Microbiology, University Hospital of Muenster, Muenster, Germany
2Molecular
Staphylococcus aureus is an important human pathogen that can cause long-lasting persistent infections. The
mechanisms by which persistent infections are maintained involve both bacterial evasion strategies and modulation of
the host immune response. So far, the investigations in this area have focused on strategies used by S. aureus to
persist within the host. We used an experimental mouse model to investigate the immune response to persistent S.
aureus infection. Our results demonstrate that T cells which were critical for controlling S. aureus infection gradually
lost their ability to respond to cognate antigenic restimulation and entered a state of anergy with the progression of
the infection towards persistence. The unresponsiveness of T cells could be reverted by additional stimulation with
the phorbol ester PMA, an activator of protein kinase C, suggesting that a defect in co-stimulation underlay the
anergic phenotype. The development of T cell anergy may contribute to the failure of the host immune response to
promote sterilizing immunity during persistent S. aureus infections but also offers the possibility for novel
immunotherapeutic approaches.
“Interleukin (IL)-4/ IL-13 receptor distribution on immune cells following vaccinia virus
infection: implications on CD8+ T cell functionality”
D. K. Wijesundara1, D. C. Tcharke2, R. J. Jackson1, C. Ranasinghe1
1Immunology,
2Biomedical
The John Curtin School of Medical Research, Acton, Australia
Science and Biochemistry, The Research School of Biology, Acton, ACT, Australia
We have previously reported that interleukin (IL)-4 and IL-13 play an important role in modulating CD8+ T cell avidity,
but the importance of IL-4/IL-13 signaling receptors in modulation of CD8+ T cell functionality is poorly understood in
the context of virus infections. To address this we used vaccinia virus (VV) as a model infection system. Our data
suggest that IL-4Ra unlike other IL-4/IL-13 receptor components was significantly down-regulated on CD8+ T cells of
BALB/c mice as a consequence of vaccinia infection. Kinetics analysis showed that IL-4Ra down-regulation in this
instance was time-dependent and correlated extremely well with the emergence of vaccinia-specific effector CD8+ T
cells. Using various functionality assays, we found that CD8+ T cells that expressed low levels of IL-4Ra (IL-4Ralo cells)
harbored vast majority (~80%) of the total VV-specific effector CD8+ T cells during the peak of the effector response.
VV infection of congenic C57BL/6.SJL mice that have received C57BL/6 OT-I splenocytes via adoptive transfer
demonstrated that IL-4Ra was selectively down-regulated on vast majority of the endogenous effector CD8+ T cells,
but not on the bystander OT-I CD8+ T cells. Furthermore, IL-4Ra down-regulation on effector CD8+ T cells was also
observed in BALB/c mice infected with A/PR8 influenza, modified vaccinia Ankara and fowl pox virus suggesting that
this phenomena is not VV specific. Infection of gene-knock out mice for IL-4, IL-13, transcription factor STAT6 and IFNg with VV confirmed that these factors were not critical for the observed reduction of IL-4Ra on effector CD8+ T cells.
Overall, our data reveal a novel phenotype of virus-specific effector CD8+ T cells where IL-4Ra expression is downregulated on effector CD8+ T cells, but not on bystander CD8+ T cells. We are currently in the process of
understanding the mechanisms involved in reduction of IL-4Ra on CD8+ T cells.
S e s s i o n I I: Im m u n e r e s p o n s e t o b a c t e r i a l p a t h o ge n s
10.45 AM – 12.35 PM
“Potential novel therapeutic strategies for control of allergic airways disease”
D. Strickland
Telethon Institute for Child Health Research
Asthma is a chronic inflammatory disease affecting mainly small/central airways. In the most common form of this
disease, allergic/atopic asthma, the underlying mechanisms involve exaggerated immune responses to aeroallergens,
including a major Th2-associated component. However <25% of sensitized subjects develop significant asthma,
suggesting that mechanisms downstream of allergic sensitization, presumably involving control of expression of Th2
immunity in airway tissue, play a central role in the disease.
In this context an increasingly important focus of recent studies on asthma pathogenesis has been the endogenous
control mechanisms that modulate immune inflammatory processes in the airway mucosa. Of particular interest are T
regulatory (Treg) populations that control T-effector responses and can effectively resolve airways
hyperresponsiveness, and deficiencies in capacity to generate mucosal-homing Tregs are widely believed to be
important determinants of asthma risk. The potential relevance of these cells as drug targets is supported by indirect
evidence from a variety of association studies demonstrating induction of Treg during various treatment protocols
that attenuate asthmatic responses, but objective proof-of-concept data with linked mechanistic evidence is lacking,
and this represents the focus of our current investigations.
“Role of IDO and tryptophan catabolism in chronic mycobacterial disease pathogenesis”
K. M. Plain1, K. De Silva1, J. Earl2, D. J. Begg1, A. C. Purdie1, R. J. Whittington1
1
Farm Animal and Veterinary Public Health, University of Sydney, Camden, NSW, Australia
Dept., The Children's Hospital at Westmead, Westmead, NSW, Australia
2Biochemistry
This study examined immune regulatory pathways involved in mycobacterial disease pathogenesis. Indoleamine 2,3dioxygenase (IDO) regulates tryptophan metabolism and was originally reported to have a role in intracellular
pathogen killing. It has been shown to be a potent immunoregulatory molecule, particularly in chronic immune
diseases. Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal disease in cattle and
sheep and an association with Crohn's disease in humans is under investigation. MAP infection progresses slowly with
a subclinical phase that leads to clinical disease in a proportion of cases. Using an experimental infection model, we
examined changes that occurred throughout the disease process from subclinical through to clinical disease at the
level of gene expression, protein localisation and also functional effects by HPLC determination of plasma tryptophan
levels. IDO gene expression was found to be increased in peripheral blood cells of MAP-exposed sheep and cattle.
MAP-infected monocytic cells had significantly increased IDO mRNA levels and both IDO gene and protein expression
were significantly increased within the tissues of affected sheep. This was particularly evident at the site of primary
infection (ileum) of animals with severe multibacilliary disease. IDO breaks down tryptophan and the increased IDO
was functional as shown by decreases in plasma tryptophan levels that correlated with the onset of clinical signs, a
stage associated with Th1 immunosuppression. A novel pathway in mycobacterial infections by which the pathogen
may harness host immune regulatory pathways to aid survival has been described, involving IDO production and
tryptophan catabolism. These findings raise new questions about the host:mycobacteria interactions in the
progression from subclinical to clinical disease. MAP infection in the natural host may be a useful tool that leads to
important findings broadly relevant to the pathogenesis of other virulent mycobacterial infections such as
tuberculosis.
“Immune correlates of protection and susceptibility to mycobacterial disease
pathogenesis”
K. De Silva1, D. J. Begg1, K. M. Plain1, A. C. Purdie1, S. Kawaji2, R. J. Whittington1
1
Faculty of Veterinary Medicine, University of Sydney, Camden, NSW, Australia
Institute of Animal Health, Tsukuba, Japan
2National
Mycobacterial diseases such as tuberculosis and paratuberculosis continue to be of serious health and economic
concern to humans and livestock globally. Disease diagnosis and control are difficult as they are slow growing
organisms causing slow progressing disease with active and latent phases. Lack of knowledge on the nature of the
protective immune response as well as immunological markers of disease susceptibility and latency hamper the
development of effective vaccines and other therapies. Unlike murine models, the use of a ruminant infection model
enabled us to follow the host response to mycobacterial challenge from the time of exposure until manifestation of
clinical disease in a natural host. Using a well-characterised experimental infection model in sheep, this study (a total
of 30 controls and 58 challenged in two trials) tracked cellular and humoral responses, as well as quantity of
mycobacterial shedding, for up to 30 months post exposure. Infection was defined as the presence of viable organisms
in tissue sections taken at necropsy. The IFNγ response increased early after exposure regardless of disease outcome.
Infected animals with lesions containing abundant mycobacteria could be distinguished from animals that developed
less severe disease. Their IL-10 response remained relatively unchanged throughout the course of disease while IL-10
levels increased with time in animals with less severe disease. Also, from early on (4 months post exposure), the
amount of mycobacterial DNA shed by animals with severe lesions was higher than in other diseased animals.
Interestingly, the cellular immune response in animals with these severe lesions showed a similar pattern to animals
with no lesions, though disease outcomes were vastly different. Exposed animals showing no signs of clinical disease
had a stronger lymphoproliferative response than those with clinical disease and continued to do so at more than 2
years after exposure. These studies demonstrate the complexity of the immune response to pathogenic mycobacteria
and illustrate the fact that measurement of a single immunological parameter is insufficient to determine the
outcome of disease.
“The deadly duo: how a tropical infection decimates a diabetic host”
J. L. Morris1, K. A. Hodgson1, N. L. Williams1, C. Rush1, B. L. Govan1, K. Sangla2, R. E. Norton3, N. Ketheesan1
1Microbiology
& Immunology, School of Veterinary and Biomedical Sciences, James Cook University, Townsville, QLD, Australia
Department of Endocrinology, Townsville Hospital, Townsville, QLD, Australia
3Pathology Queensland, Townsville Hospital, Townsville, QLD, Australia
2
Gram-negative bacterial infection is a significant cause of sepsis. In the tropics, Burkholderia pseudomallei, which
causes melioidosis is a major contributor to community-acquired sepsis. In the past two years there has been a three
fold increase in cases of melioidosis in Australia. Septicaemic melioidosis is associated with mortality rates of up to
80%. Since more than half of patients with melioidosis also have type 2 diabetes (T2D) our studies have focused on
determining the host factors responsible for causing the rapid progression in this population. We used a human whole
blood model of T2D-melioidosis co-morbidity together with animal models of co-morbidT2D and melioidosis to
investigate early host-pathogen interactions that contribute to increased susceptibility.
Following exposure to B. pseudomallei, differences were observed in expression of TLR2, CD14 and CD11b on
phagocytes and in IL-12p70, MCP-1 and IL-8 plasma levels in whole blood from individuals with T2D compared to nondiabetic individuals. Data from these studies demonstrate that very early interactions between the pathogen and
phagocytes are significantly altered in hosts with T2D. This is supported by studies utilising our animal models.
We found baseline expression of IL-1β (0.07vs0.24; P=0.03) and TNF-α (0.40vs1.15; P=0.02) was significantly higher in
subcutaneous adipose tissue of T2D mice and that these mice were significantly more susceptible to both intranasal
and subcutaneous infection. T2D mice with extended periods of uncontrolled hyperglycaemia had impaired DC and
macrophage function. Despite similar organ bacterial loads at day 1 post-infection, cytokine expression and tissue
pathology was exacerbated in T2D mice with the hyperinflammatory response leading to overwhelming sepsis and
increased mortality by day 3 post-infection.
Our observations prove that antimicrobial therapy alone may be futile in facilitating the recovery of the patient and
that the therapeutic regime should include agents aimed at ameliorating the early events that lead to the cytokine
storm.
“NK cell-derived IFNγ contributes to pathology in a murine model of Streptococcus
pneumoniae meningitis”
A. J. Mitchell1, B. Yau1, L. Too1, J. A. McQuillan1, H. J. Ball1, C. Jones3, I. L. Campbell2, P. J. Hertzog4, N. H.
Hunt1
1Sydney
Medical School, University of Sydney, Camperdown, NSW, Australia
of Molecular and Microbial Biosciences, University of Sydney, Camperdown, NSW, Australia
3
Children's Hospital Westmead, University of Sydney, Westmead, NSW, Australia
4Centre for Innate Immunity & Infectious Disease, Monash Institute of Medical Research, Clayton, VIC, Australia
2School
Bacterial meningitis is a major cause of morbidity and mortality world wide. Using a murine model of Streptococcus
pneumoniae meningitis we investigated the influence of the host immune response on the development of pathology.
Array analysis of gene expression in the brains of infected mice showed that of the top 200 differentially expressed
genes, 159 were classifiable as either type-I or type II interferon dependent. Despite this, induction of type I interferon
mRNA species was unable to be demonstrated, nor were IFNARI or IFNAR2 gene knockout (KO) mice protected from
pathology. Conversely, both IFNγ mRNA and IFNγ protein were highly upregulated in the brains of infected mice, and
IFNγ KO were significantly protected from morbidity, with 73% of mice surviving >200 hours compared to a median
survival of 88 hours in wild-type animals. Despite protection in IFNγ KO mice, the exact IFNγ-dependent processes
driving pathology have not yet been identified – no evidence was found supporting a range of potential IFNγdependent pathways (cytokine gene expression, leukocyte infiltration, macrophage scavenger gene expression and
bacterial clearance, tryptophan metabolism, brain oedema, astrocyte activation and CXCR3-dependent mechanisms).
In contrast, the pathways leading to induction of IFNγ were more readily resolved. Upregulation of IFNγ was IL-18
dependent, and intracellular cytokine staining on leukocyte populations isolated from infected brains showed that the
major IFNγ producing population was NK1.1+, NKp46+ NK cells. This was confirmed by depletion of NK cells by
administration of anti-asialo-GM1 antibody, which reduced IFNγ mRNA induction >10-fold. Taken together with other
evidence, we suggest a model involving initial inflammasome formation stimulated by S. pneumoniae infection, which
leads to activation of IL-18 and subsequent production of IFNγ by NK cells. This NK cell-derived IFNγ drives
pathological processes that are still under investigation.
“SSL7 – S. aureus’s answer to the complement system”
N. Lorenz, F. Clow, F. Radcliff, J. Fraser
Infection & Immunity, School of Medical Sciences, University of Auckland, Auckland, New Zealand
Staphylococcus aureus is a major cause of severe hospital- and community-acquired infections. S. aureus generates
numerous virulence factors known to interfere with host immune defences. Staphylococcal superantigen-like (SSL)
proteins comprise a family of molecules with close structural homology to superantigens but lack non-specific T-cell
activation. Many SSL proteins target key components of the innate immune system and promote host colonization
and immune evasion. SSL7 binds simultaneously to IgA and complement component 5 (C5) forming a large tetrameric
complex that prevents cleavage of C5 and recognition of IgA. SSL7 is a powerful inhibitor in vitro of serum
(complement mediated) haemolytic and bactericidal activity. Additionally SSL7 eliminates end-stage complement
activation and generation of both anaphylotoxin C5a and the Membrane Attack Complex (MAC).
In a murine model of peritonitis, SSL7 potently inhibited the chemotaxis of neutrophils to the peritoneum in response
to an inflammatory stimulus. Mutagenesis of the C5 binding site on SSL7 abolished inhibition whereas mutation of the
IgA binding site had no effect. To investigate the virulence mechanism of SSL7, the ssl7 gene was expressed in the
gram-positive, avirulent Lactococcus lactis. Expression of SSL7 dramatically enhanced survival in a human whole blood
killing assay, most likely at the level of complement opsonisation consistent with SSL7's ability to inhibit the activation
of C5. However end-stage complement, is considered downstream of the main opsonins C4b and C3b. This potentially
highlights a previously unknown role for C5b in opsonophagocytosis. SSL7 is proving to be a powerful tool to dissect
the role of complement in cell mediated killing of gram-positive organisms. A deeper understanding of the
interactions of virulence factors and host defence may provide the basis for new strategies in the treatment of
bacterial infections.
S e s s i o n I II : In f l a m m a t i o n a n d D i s e a s e
1:30 PM – 2.35 PM
“Tolerance, Tissue Defence and the Th17 response”
R. Flavell
Yale University
The Th 17 response protects against infection but causes severe immunopathology. How is the balance between these
events maintained? We find that Th17 cells are diverted to the small intestine where they are tolerized by IL-10. The
mechanisms which underlie this process, the source of IL-10 and the lineage relationships which prevail in this system
will be discussed.
“Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a
mechanism for enhanced IL-1Beta in type 2 diabetes”
S. L. Masters1,2
1Immunology
Research Centre
Inflammation Research Group, School of Biochemistry and Immunology, Trinity College Dublin, Ireland
2
IL-1β is an important inflammatory mediator of type 2 diabetes (T2D), and preventing the action of this cytokine
benefits patients suffering from the disease. Here we show that oligomers of islet amyloid polypeptide (IAPP), a
protein that forms amyloid deposits in the pancreas during T2D, can activate the Nlrp3 inflammasome and thus
generate mature IL-1β. This occurs in macrophages, which have been found in the pancreas to engulf IAPP but cannot
degrade the amyloid normally. A T2D therapy, glyburide, as well as inhibitors targeting reactive oxygen species
production, and cathepsin B activation, prevented IAPP-mediated IL-1β secretion in vitro. Surprisingly a recently
identified Nlrp3 ligand that is implicated in T2D, Txnip, is not required for inflammasome activation by IAPP, and is not
downregulated by glyburide in macrophages. The effect of IAPP on IL-1β first required priming of the inflammasome, a
step that we have found can be carried out by minimally oxidized low density lipoprotein. Furthermore we observed
that glucose metabolism was required for priming to proceed, suggesting why hyperglycemia in T2D may be
associated with increased IL-1β. Finally, mice transgenic for human IAPP deposit amyloid in pancreatic islets, and this
decreased the number of insulin producing cells while increasing the expression of IL-1β. There is also a high degree of
co-localization with macrophages and amyloid staining in the regions where IL-1β is present. Our findings elucidate a
novel mechanism for IL-1β production in T2D and may provide new avenues for pharmacological intervention in
pathology caused by IAPP.
S e s s i o n IV : M u c o s a l Im m u n o l o g y
2:50 PM – 4:30 PM
“Generation and function of immunosuppressive CD4+ and CD8+ FoxP3+ regulatory T
cells in the intestinal mucosa”
D. Fleissner1, M. Knott1, T. Knuschke1, S. Jung2, R. Geffer3, W. Hansen1, J. Buer1, A.M. Westendorf1
1Institute
of Medical Microbiology, University Hospital Essen, University Duisburg-Essen, Germany
of Immunology, Weizmann Institute of Science, Rehovot, Israel
3
Department of Cell Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany
2Department
Immune responses in the intestinal tract are different from those in peripheral lymphoid organs. The immunologic
tone of the gut-associated lymphoid tissue is one of suppression rather than of active immunity. Therefore, the
intestinal immune system has evolved redundant regulatory mechanisms to prevent unwarranted inflammation. This
involves regulatory T cells residing in large numbers in the gut. We hypothesized that the gut environment
preferentially supports unique ways to expand these cells.
First, in a transgenic mouse model we demonstrated that intestinal self-antigen expression leads to peripheral
expansion of antigen-specific CD4+Foxp3+ regulatory T cells. Although gut-associated dendritic cells are able to induce
self-antigen-specific CD4+Foxp3+ T cell proliferation, in vivo depletion of dendritic cells did not preclude the expansion
of these regulatory T cells in vivo. Importantly, we found that self-antigen presentation by primary intestinal epithelial
cells (IECs) is also sufficient to expand self-antigen specific CD4+Foxp3+ regulatory T cells. This mechanism is
dependent on MHC class II but unlikely to be dependent on TGF-β and retinoic acid, mediators that are abundantly
produced in the intestinal mucosa and discussed in the context of CD4 + regulatory T cells. Our study provides
experimental evidence for a new concept in mucosal immunity, that expansion of CD4 + regulatory T cells can be
achieved independent of local DCs through antigen-specific IEC – T cell interactions.
Second, in contrast to CD4+Foxp3+ regulatory T cells, immunomodulatory CD8+ T cells that express Foxp3 have not
been well defined in terms of their generation and function. In a transgenic mouse model of intestinal tolerance we
identified a population of CD8+Foxp3+ T cells that exert an immunosuppressive effect on CD8+ and CD4+ T cells in vitro.
Interestingly, the frequency of CD8+Foxp3+ T cells is reduced in the peripheral blood of patients with ulcerative colitis.
As these cells might play yet an underestimated role in the maintenance of intestinal homeostasis, we have
investigated human and murine CD8+Foxp3+ T cells generated by stimulating naïve CD8+ T cells in the presence of TGFβ and retinoic acid. These CD8+Foxp3+ fully competent regulatory T cells show strong expression of regulatory
molecules e.g. CD25, Gpr83 and CTLA-4, exhibit cell-cell contact–dependent immunosuppressive activity in vitro and
interfere with inflammation in vivo. Thus, our study illustrates a previously unappreciated critical role of CD8+Foxp3+ T
cells in controlling intestinal homeostasis.
“Lung localised CD4 T cells are sufficient for protective immunity against helminth
infection in the absence of T cell recirculation”
M. C. G. Harvie1,2, S. Tang2, M. Camberis2, V. Brinkmann3, H. Mearns2, G. Le Gros2
1Institute
of Health and Biomedical Innovation (IHBI), Queensland University of Technology, Kelvin Grove, Australia
Malaghan Institute of Medical Research, Wellington, New Zealand
3Novartis Institutes for Biomedical Research, Basel, Switzerland
2
A well-developed understanding of protective immunity is vital for the design of effective vaccines and treatments.
Secondary lymphoid based immune responses are known to be important in the development of protective immunity,
however the role of peripheral tissue immune responses remains unclear. This is an important issue particularly in
relation to the development of vaccines targeted at mucosal surfaces. Using the drug FTY720 to restrict lymphocyte
recirculation, we assessed the roles of lung and lymph node based CD4 Th2 cell responses in the development of
protective immunity against Nippostrongylus brasiliensis. Strikingly, we found that CD4 T cell responses localized in
the lung tissue could protect against N. brasiliensis infection. This finding suggests that peripheral tissue adaptive
immune responses play an important role in controlling infection, with tissue localized cells responding rapidly upon
reinfection to confer protection.
“Protection against respiratory bacterial infection by oral immunisation”
M. L. Dunkley1,2, R. L. Clancy1,2,3
1R&D
Unit, Hunter Immunology Ltd, Callaghan, Australia
of Biomedical Sciences and Pharmacy, Faculty of Health, University of Newcastle, Newcastle, NSW, Australia
3
Hunter Area Pathology Service, John Hunter Hospital, Newcastle, NSW, Australia
2School
Oral immunization of rats with killed P. aeruginosa results in enhanced bacterial clearance of a subsequent respiratory
infection with live P. aeruginosa. This is associated with activation of airway macrophages and enhanced recruitment
and activation of polymorphonuclear neutrophils (PMN). The role of antigen-specific CD4+ T lymphocytes in enhanced
phagocytosis and bacterial killing was demonstrated in vitro where mesenteric lymph node (MLN) CD4+T cells from
immunized or unimmunized rats were mixed with live P. aeruginosa and PMN or macrophages isolated from nonimmune rat blood or airways respectively.
Studies of oral immunization with killed non-typeable Haemophilus influenzae (NTHi) in rodent models have
demonstrated similar protection against subsequent NTHi respiratory infection challenge, and protection was
associated with induction of specific T cells that secrete IFNγ and IL-17. In human studies of oral immunization with
enteric-coated tablets containing killed NTHi, induction of NTHi-specific T cells and reduction in NTHi and other
bacterial species (P. aeruginosa, S. pneumonia, M. catarrhalis) in the airways was apparent when compared to the
placebo-treated patient group.
“Effects of milk and soy protein-based diets on caecal microbiota profiles in the
Interleukin-10 gene-deficient mouse”
A. J. Hodgkinson1, W. Young2, N. A. McDonald1, M. P.G. Barnett2, C. G. Prosser4, N. C. Roy2,3
1Food
and Bio-based Products, AgResearch Ltd, Hamilton, New Zealand
Food and Bio-based Products, AgResearch Ltd, Palmerston North, New Zealand
3
Riddet Institute Massey University, Palmerston North, New Zealand
4Dairy Goat Co-operative (NZ) Ltd, Hamilton, New Zealand
2
Gastrointestinal homeostasis is maintained by interactions between host, microbiota and diet. If the balance is
disturbed, inflammatory disease can result. Interleukin-10 (IL-10) plays a pivotal immuno-regulatory role in
suppressing inflammatory processes. We compared effects of soy, cow milk or goat milk protein-based diets on caecal
microbiota profiles in Il10 gene-deficient mice, a model of intestinal inflammation.
Five week-old Il10 – / – ( n = 15/group) and C57BL/6J (Control; n = 8/group) male mice sourced from a SPF laboratory,
were assigned to 3 treatment groups randomized by weight. Mice were inoculated with endogenous intestinal
bacteria (including Enterococcus strains) to normalise gastrointestinal microbiota. They were then fed diets (modified
AIN-76A) containing 20% protein from soy, cow milk or goat milk for 6 weeks with body weight and condition
monitored. At 11 weeks, mice were euthanized and samples collected.
Il10 – / – mice fed soy-based diet gained weight similar to C57BL/6J control mice. In contrast, the Il10 – / – mice fed milkbased diets lost weight and developed diarrhoea. Acute phase protein (SAA) plasma levels were 295, 85 and 25 times
higher for cow milk, goat milk and soy protein-based diets, respectively, compared with C57BL/6J controls. Milk-based
diets induced greater expression of inflammatory genes in Il10 – / – mice compared with soy-based diet. Although all
animals had been dosed with the same endogenous intestinal bacteria, evaluation of caecal microbiota profiles by
TTGE showed a clear distinction between Il10 – / – mice and C57BL/6J mice. Whilst in C57BL/6J control mice there was
no effect of dietary treatment, in Il10 – / – mice there appeared to be differentiation in microbiota profiles between soy
and milk-based diets. Comparison of physical, biochemical and gene expression data suggest that soy protein-based
diets ameliorate development of intestinal inflammation in Il10 – / – mice in contrast to milk-based diets. These
differences may be due in part to variation in their microbiota profiles.
“Role of novel type I Interferon Epsilon (IFN-ε) in viral infection and mucosal immunity”
Y. Xi, S. L. Day, R. J. Jackson, C. Ranasinghe
Immunology, John Curtin School of Medical Research, Acton, ACT, Australia
The role of interferon epsilon (IFN-ε) in mucosal immunity was evaluated utilizing recombinant viral vectors expressing
IFN-ε. Intranasal (i.n.) VV-HIV-IFN-ε infection, induced heightened vaccinia-specific IFN-γ+CD107a+CD8+ T cells in the
lung with enhanced activation markers CD69 and CD103. Moreover, a highly unusual CD8+CD4+ double positive (DP)
CD107a+IFN-γ+ T cell subset was detected in lung lymph nodes. These factors correlated with a rapid lung VV
clearance. Following i.n./intramuscular (i.m.) FPV-HIV-IFN-ε/VV-HIV-IFN-ε prime-boost immunization, co-expression of
IFN-ε enhanced HIV-specific T cell responses in spleen, genito-rectal nodes and Peyer's patches, compared to control
vaccination, but did not alter T cell avidity. Independent of the route of delivery IFN-ε induced elevated
KdGag+CD8+α4β7+CCR9+ T cells in Peyer's patches suggesting that IFN-ε can promote migration of T cells to the gutmucosae. Our observations suggest that IFN-ε could serve as an antiviral agent to prevent or reduce mucosally related
infections in the lung and gut, where it could act as a first line of defence against diseases such as TB and HIV-1.