Cathryn Nagler, Ph.D.

Tolerance and immunity in the intestinal mucosa; the role of innate immune signals in regulation of allergic and inflammatory disease; mucosal vaccines

Research Summary

How do immune cells in  the intestinal mucosa distinguish innocuous dietary antigens and trillions of  commensal bacteria from pathogenic microbes and mount an appropriate response  to each? Our  laboratory is pursuing several different, but complementary, lines of research aimed at examining the mechanisms regulating non-responsiveness to these stimuli in healthy individuals and its abrogation in food allergy and inflammatory bowel disease. Our murine disease models will provide the pre-clinical basis for future translational studies aimed ultimately at the development of novel immunotherapeutic modalities.

Our work on tolerance to dietary antigen has shown thatchronic enteric helminth infection acts as an adjuvant to prime for a Th2 biased cell-mediated and antibody response to an otherwise tolerogenic oral administration of a model dietary antigen.  Allergens and helminth infection are the two types of stimuli known to uniquely induce Th2 biased responses and the production of IgE.  However, instead of enhanced allergic hyperreactivity to dietary antigens in helminth-infected mice, we found that Th2 responses occurred without the development of atopy. In both our murine model and in epidemiological studies in the developing world, enteric helminth infection protects against allergic disease. Protection correlates with the helminth’s ability to induce a modified Th2 response, characterized by the production of Th2 cytokines in the context of high levels of immunomodulatory IL-10 and increased numbers of regulatory T cells. The immunomodulatory features of helminth infection have also been shown to be effective in controlling intestinal inflammation in both murine models and in clinical trials of patients with inflammatory bowel disease. Current work in our laboratory (and others) is focused on identifying the microbial constituents responsible for helminth-induced immunomodulatory activity at the molecular level and characterizing immunoregulatory antigen presenting cell and regulatory cell populations in detail.

Environmental influences are implicated in the rapidly rising incidence of food allergy. We have found that mice which are unable to signal via TLR4, the receptor for bacterial LPS, are highly susceptible to an allergic response to food. When the composition of the microbiota is reduced and altered by antibiotic treatment, TLR4 wild type mice develop allergic responses similar to their TLR4 mutant counterparts. More recent work has shown that IL-10 secreting regulatory dendritic cells (DC) and T cells present in the draining mesenteric lymph node (MLN) of wild type mice are absent from the MLN of both TLR4 mutant mice and antibiotic-treated WT mice. Our data therefore suggests that TLR4 mutant mice are highly susceptible to allergic responses to food antigens because they lack populations of microbiota induced regulatory DC and T cells which are present in wild type mice. We are currently examining which mucosal cell populations initially respond to the bacterial signals, and how this signal is transmitted to the draining lymph node where antigen presentation takes place. We will also examine the role of various subcomponents of the commensal microbiome in the regulation of allergy, with a focus on the Helicobacter species that infect large segments of the population.

We initially prepared TLR4-/- x IL-10-/- (DKO) mice to study the role of IL-10 in the regulation of allergic responses to food in TLR4 mutant mice. We found that spontaneous Helicobacter-dependent colitis was exacerbated in IL-10-/- mice that bear a mutation in TLR4. TLR4 mediated signals play two interrelated roles in the exacerbation of disease in TLR4-/- x IL-10-/- mice. Both proinflammatory and immunoregulatory cytokine secretion is dysregulated; IFN-y and IL-17 secreting Foxp3+ Tregs accumulate in the colonic LP of TLR4-/- x IL-10-/- mice and fail to control disease. Aberrant control of epithelial cell turnover results in the persistence of antigen presenting cells bearing apoptotic epithelial fragments in the colonic lamina propria of Helicobacter infected TLR4-/- mice. Our data suggests that, in TLR4-/- mice, IL-10 secretion by these APC prevents an inflammatory response. In mice that lack both IL-10 and TLR4-mediated signals, aberrant regulatory cell function and dysregulated control of epithelial homeostasis combine to exacerbate disease.

As another approach to understanding the mechanisms regulating tolerance to dietary antigen, we examined the response to the same model antigen (ovalbumin, OVA) presented orally in a tolerogenic (soluble) and an immunogenic (vaccine) form.  We created a novel, clinically relevant attenuated Salmonella vaccine in which OVA is expressed under the control of an anaerobically inducible promoter suitable for the harsh gastrointestinal microenvironment. To gain insight into the role of innate immune signals in the regulation of tolerance we administered the two types of antigen to mice deficient in the TLR adaptor protein MyD88.  Although some earlier work had suggested that MyD88 signaling was required for the generation of an antibody response, we found that vaccination of MyD88 -/- mice with Salmonella-OVA induced robust serum antibody responses in the absence of MyD88 signaling that were predominantly comprised of Th2 and TGF-b dependent serum antibody isotypes. MyD88-mediated signals were also required for CD1d-restricted invariant natural killer (iNK)T cell activation following oral vaccination with Salmonella-OVA.   Our data suggest that in the absence of MyD88, the route of immunization governs the magnitude and type of antibody response elicited.  Moreover, in wild type mice, activated, CD1d-restricted invariant iNKT cells are required for an optimal Th1-dependent antigen-specific serum antibody response to an orally administered Salmonella vaccine.

The potent immunomodulatory properties of intestinal helminth infection also present a challenge to effective vaccination in the developing world.  In recent work we have found that chronic enteric helminth infection impairs the serum antibody response to a protein antigen adsorbed to alum and administered by intramuscular injection (as a model for the form of antigen that serves as the basis for nearly all currently licensed human vaccines).  Helminth infection also impairs the antibody response to the same antigen expressed using the attenuated Salmonella strain as a mucosal vaccine vector. Although currently little studied, our findings make clear that strategies to counter the ability of endemic helminth infection to modulate the response to both of these commonly used forms of vaccination will be an important consideration for future vaccines designed for implementation in the developing world.

Selected Papers

Shi, H.N., Ingui, C. J., Dodge, I. and C. Nagler-Anderson.  A helminth induced mucosal Th2 response alters non-responsiveness to oral administration of soluble antigen.  J. Immunol. 1998;160:2449-2455.

Spiekermann, G.M. and C. Nagler-Anderson.  Oral administration of the bacterial superantigen Staphylococcal enterotoxin B induces activation and cytokine production by T cells in murine gut associated lymphoid tissue. J. Immunol. 1998; 161: 5825-5831.

Shi, H.N., Grusby, M.J. and C. Nagler-Anderson.  Orally induced peripheral non-responsiveness is maintained in the absence of functional Th1 or Th2 cells.  J. Immunol. 1999; 162: 5143-5148.

Fox, J.G., Beck, P., Dangler, C.A., Whary, M.T., Wang, T.C., Shi, H.N. and C. Nagler-Anderson.  Concurrent enteric helminth infection modulates inflammation, gastric immune responses, and reduces Helicobacter-induced gastric atrophy.  Nature Medicine 2000; 6: 536-542.

Shi, H.N., Liu, H.Y. and C. Nagler-Anderson.  Enteric infection acts as an adjuvant for the response to a model food antigen, J. Immunol., 2000, 165: 6174-6182.

Nagler-Anderson, C., Terhorst, C., Bhan, A.K. and Podolsky D.K. Mucosal antigen presentation and the control of tolerance and immunity.  Trends in Immunology 22; 2001, 120-122.

Azuara, V., Grigoriadou, K., Lembezat, M.-P., Nagler-Anderson, C. and P. Pereira.  Strain-specific TCR repertoire selection of IL-4 producing Thy-1 dull gd thymocytes. Eur. J. Immunol, 2001; 31: 205-214.

Nagler-Anderson, C.  Man the barrier! Strategic defences in the intestinal mucosa.  Nature Reviews Immunology 1; 2001, 59-67.

Bashir, M.E.H., Andersen, P., Fuss, I.J., Shi, H.N. and C. Nagler-Anderson.  An enteric helminth infection protects against an allergic response to dietary antigen.  J. Immunol. 2002, 169:3284-3292.

Nagler-Anderson, C., Bhan, A.K., Podolsky, D.K. and C. Terhorst.  Control freaks: immune regulatory cellls. Nature Immunol. 2004, 5: 119-122.

Bashir, M.E.H., Louie, S., Shi, H.N. and C. Nagler-Anderson.  TLR-4 signaling by intestinal microbes influences susceptibility to food allergy.  J.Immunol., 2004, 172, 6978-6987.

Smith, D.W. and C. Nagler-Anderson.  Preventing intolerance: the induction of non-responsiveness to dietary and microbial antigens in the intestinal mucosal.  J. Immunol. 2005, 174: 3851-3857.

Iweala, OI and C. Nagler   Immune privilege in the gut:  the establishment and maintenance of non-responsiveness to dietary antigens and commensal flora.  Immuno. Rev. 2006, 213: 82-100.

Maillard, M.H., Cotta-de-Almeida, V., Takeshima, F., Nguyen, D.D., Michetti, P., Nagler, C. Bhan, A.K. and Snapper, S.B.  The Wiskott Aldrich Syndrome Protein is required for the function of CD4+CD25+Foxp3+ regulatory T cells. J. Exp. Med. 2007, 204: 381-391.

Urban, J.F., Jr. Steenhard, N.R., Solano-Aguilar, G.I., Dawson, H.D., Iweala, O.I, Nagler, C.R., Noland, G.S., Kumar, H., Anthony, R.M., Shea-Donohue, T, Weinstock, J. and Gause, W.  Infection with parasitic nematodes confounds vaccine efficacy. Vet. Parasitol. 2007, 148: 14-20.

Nguyen, D.D., Maillard, M.H., Cotta-de-Almeida, V., Mizoguchi, E., Klein, C., Fuss, I. Nagler, C., Mizoguchi, A., Bhan, A.K. and Snapper, S.B. Lymphocyte-dependent and Th2 cytokine associated colitis in mice deficient in Wiskott-Aldrich Syndrome protein.  Gastroenterol. 2007. 133: 1188-1197.

Harris JB, Podolsky MJ, Bhuiyan TR Chowdhury F, Khan AI, LaRocque RC, Logvinenko T, Kendall J, Faruque AS, Nagler C, Ryan ET, Qadri F, Calderwood SB. Immunologic responses to V. cholerae in patients co-infected with intestinal parasites in Bangladesh. PLoS Negl Trop Dis, 2009; 3(3):e403. Epub 2009 Mar 31.

Iweala, O.I., Smith, D.S., Matharu, K.S., Sada-Ovalle, I., Nguyen, D.D., DeKruyff, R.H., Umetsu, D.T., Behar, S.M and C. R. Nagler.  Vaccine induced antibody isotypes are skewed by impaired CD4 T cell and invariant NKT cell effector responses in MyD88 deficient mice, Journal of Immunology, 2009,183:2252-2260.

Matharu, K.S, Mizoguchi, E., Alonso,  C., Nguyen D., Mingle, B., Iweala,  O.I., McBee, M. Stefka, AT, Prioult, G, Haigis, K.M., Bhan,  A.K., Snapper, S.B., Murakami, H, Schauer D.B. Reinecker,H.C Mizoguchi, A. and C. R. Nagler.  TLR4-mediated regulation of spontaneous Helicobacter-dependent colitis in IL-10  deficient mice. Gastroenterology  2009, 137: 1380-1390

McBee M.E., Zeng Y, Parry N, Nagler C.R., Tannenbaum S.R. and D.B. Schauer. Multivariate modeling identifies neutrophil- and Th17-related factors as differential serum biomarkers of chronic murine colitis. PLoS ONE 2010, 5: e13277.

Wang, Y., Devkota, S., Musch, M.W., Jabri, B., Nagler, C., Antonopoulos, D.A., Chervonsky, A and E.B. Chang. Regional mucosa- associated microbiota determine physiological expression of TLR2 and TLR4 in murine colon.  PLoS ONE 2010, 5: e13607.