-
[show abstract]
[hide abstract]
ABSTRACT: Salmonella enterica serovar Typhimurium is a clinically important gram-negative, enteric bacterial pathogen that activates several Toll-like receptors (TLRs). While TLR signaling through the adaptor protein MyD88 has been shown to promote inflammation and host defense against the systemic spread of S. Typhimurium, curiously, its role in the host response against S. Typhimurium within the mammalian gastrointestinal (GI) tract is less clear. We therefore used the recently described Salmonella-induced enterocolitis and fibrosis model: wild-type (WT) and MyD88-deficient (MyD88(-/-)) mice pretreated with streptomycin and then orally infected with the ΔaroA vaccine strain of S. Typhimurium. Tissues were analyzed for bacterial colonization, inflammation, and epithelial damage, while fibrosis was assessed by collagen quantification and Masson's trichrome staining. WT and MyD88(-/-) mice carried similar intestinal pathogen burdens to postinfection day 21. Infection of WT mice led to acute mucosal and submucosal inflammation and edema, as well as significant intestinal epithelial damage and proliferation, leading to widespread goblet cell depletion. Impressive collagen deposition in the WT intestine was also evident in the submucosa at postinfection days 7 and 21, with fibrotic regions rich in fibroblasts and collagen. While infected MyD88(-/-) mice showed levels of submucosal inflammation and edema similar to WT mice, they were impaired in the development of mucosal inflammation, along with infection-induced epithelial damage, proliferation, and goblet cell depletion. MyD88(-/-) mouse tissues also had fewer submucosal fibroblasts and 60% less collagen. We noted that cyclooxygenase (Cox)-2 expression was MyD88-dependent, with numerous Cox-2-positive cells identified in fibrotic regions of WT mice at postinfection day 7, but not in MyD88(-/-) mice. Treatment of WT mice with the Cox-2 inhibitor rofecoxib (20 mg/kg) significantly reduced fibroblast numbers and collagen levels without altering colitis severity. In conclusion, MyD88 and Cox-2 signaling play roles in intestinal fibrosis during Salmonella-induced enterocolitis.
AJP Gastrointestinal and Liver Physiology 06/2012; 303(3):G311-23. · 3.43 Impact Factor
-
S Ghosh,
C Dai,
K Brown,
E Rajendiran,
S Makarenko,
J Baker,
C Ma,
S Halder, M Montero,
V A Ionescu,
A Klegeris,
B A Vallance,
D L Gibson
[show abstract]
[hide abstract]
ABSTRACT: Individuals vary in their resistance to enteric infections. The role of the intestinal microbiota in altering susceptibility to enteric infection is relatively unknown. Previous studies have identified that C3H/HeOuJ mice suffer 100% mortality during Citrobacter rodentium-induced colitis, whereas C57BL/6 mice recover from infection. The basis for their differences in susceptibility is unclear and has been mainly attributed to differences in host genetics. This study investigated the role of the intestinal microbiota in altering susceptibility to C. rodentium-induced colitis. When the feces of C57BL/6 mice were gavaged into antibiotic treated C3H/HeOuJ mice, the C57BL/6 microflora led to a complete reversal in mortality patterns where 100% of the C3H/HeOuJ mice survived infection. This protection corresponded with reduced colonic pathology and less systemic pathogen load and was associated with increased inflammatory and redox responses with reduced epithelial cell death. C3H/HeOuJ mice are normally susceptible to infection-induced dehydration due to defective expression of colonic ion transporters such as Dra, CA IV, and CA I; expression of these genes was normalized when C3H/HeOuJ mice were colonized with the C57BL/6 microflora. Together, these data reveal that the colonic microbiota play a critical role in protecting against intestinal infection by inducing proinflammatory and prooxidant responses that control pathogen load as well as ion transporter gene expression previously shown to prevent fatal dehydration. Protection of mice from lethal colitis was associated with higher levels of bacteria from Bacteroidetes. This study reveals that the microbiota is sufficient to overcome inherent genetic susceptibility patterns in C3H/HeOuJ mice that cause mortality during C. rodentium infection.
AJP Gastrointestinal and Liver Physiology 03/2011; 301(1):G39-49. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Antibodies (Abs) produced during HIV-1 infection rarely neutralize a broad range of viral isolates; only eight broadly-neutralizing (bNt) monoclonal (M)Abs have been isolated. Yet, to be effective, an HIV-1 vaccine may have to elicit the essential features of these MAbs. The V genes of all of these bNt MAbs are highly somatically mutated, and the V(H) genes of five of them encode a long (≥ 20 aa) third complementarity-determining region (CDR-H3). This led us to question whether long CDR-H3s and high levels of somatic mutation (SM) are a preferred feature of anti-HIV bNt MAbs, or if other adaptive immune responses elicit them in general.
We assembled a V(H)-gene sequence database from over 700 human MAbs of known antigen specificity isolated from chronic (viral) infections (ChI), acute (bacterial and viral) infections (AcI), and systemic autoimmune diseases (SAD), and compared their CDR-H3 length, number of SMs and germline V(H)-gene usage. We found that anti-HIV Abs, regardless of their neutralization breadth, tended to have long CDR-H3s and high numbers of SMs. However, these features were also common among Abs associated with other chronic viral infections. In contrast, Abs from acute viral infections (but not bacterial infections) tended to have relatively short CDR-H3s and a low number of SMs, whereas SAD Abs were generally intermediate in CDR-H3 length and number of SMs. Analysis of V(H) gene usage showed that ChI Abs also tended to favor distal germline V(H)-genes (particularly V(H)1-69), especially in Abs bearing long CDR-H3s.
The striking difference between the Abs produced during chronic vs. acute viral infection suggests that Abs bearing long CDR-H3s, high levels of SM and V(H)1-69 gene usage may be preferentially selected during persistent infection.
PLoS ONE 01/2011; 6(3):e16857. · 4.09 Impact Factor
-
Deanna L Gibson, Marinieve Montero,
Mark J Ropeleski,
Kirk S B Bergstrom,
Caixia Ma,
Sanjoy Ghosh,
Helen Merkens,
Jingtian Huang,
Lisa E Månsson,
Ho Pan Sham,
Kelly M McNagny,
Bruce A Vallance
[show abstract]
[hide abstract]
ABSTRACT: The roles of intestinal Toll-like receptors (TLR) in the pathogenesis of colitis are not known. TLR2 and TLR4 appear to protect against dextran sodium sulfate-induced colitis by promoting mucosal integrity, but it is not clear whether this method of protection occurs in other models of colitis. We investigated the roles of TLR2 and TLR4 and the cell types that express these receptors during infectious colitis.
We generated chimeric mice with TLR2(-/-) or TLR4(-/-) bone marrow and infected them with the bacterial pathogen Citrobacter rodentium. We assessed their susceptibility to colitis and the mechanisms of TLR-mediated mucosal integrity.
TLR2-expressing tissue resident cells prevented lethal colitis, whereas TLR4-dependent inflammatory responses of hematopoietic cells mediated intestinal damage. TLR2 expression protected against intestinal damage by maintaining epithelial barrier function and inducing expression of interleukin (IL)-11 from tissue resident cells in the muscularis mucosae, concurrent with epithelial activation of the transcription factor STAT3. Addition of exogenous IL-11 protected against the lethal colitis in TLR2-deficient mice via STAT3 activation in intestinal epithelial cells.
TLR2-dependent cytoprotective responses from tissue resident cells maintain mucosal integrity against the ultimately lethal TLR4-dependent inflammatory responses of hematopoietic cells. Whereas TLR2 protects against various noxious agents, the role of TLR4 during colitis can be either protective or damaging, depending on the stimulus. Therefore, therapeutics that reduce innate immunity (TLR2 signaling in particular) may not be beneficial to patients with colitis; they could worsen symptoms. Therapies that stimulate cytoprotective responses, like IL-11, could have benefits for patients with colitis.
Gastroenterology 10/2010; 139(4):1277-88. · 11.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Salmonella enterica is an intracellular bacterial pathogen that resides and proliferates within a membrane-bound vacuole in epithelial cells of the gut and gallbladder. Although essential to disease, how Salmonella escapes from its intracellular niche and spreads to secondary cells within the same host, or to a new host, is not known. Here, we demonstrate that a subpopulation of Salmonella hyperreplicating in the cytosol of epithelial cells serves as a reservoir for dissemination. These bacteria are transcriptionally distinct from intravacuolar Salmonella. They are induced for the invasion-associated type III secretion system and possess flagella; hence, they are primed for invasion. Epithelial cells laden with these cytosolic bacteria are extruded out of the monolayer, releasing invasion-primed and -competent Salmonella into the lumen. This extrusion mechanism is morphologically similar to the process of cell shedding required for turnover of the intestinal epithelium. In contrast to the homeostatic mechanism, however, bacterial-induced extrusion is accompanied by an inflammatory cell death characterized by caspase-1 activation and the apical release of IL-18, an important cytokine regulator of gut inflammation. Although epithelial extrusion is obviously beneficial to Salmonella for completion of its life cycle, it also provides a mechanistic explanation for the mucosal inflammation that is triggered during Salmonella infection of the gastrointestinal and biliary tracts.
Proceedings of the National Academy of Sciences 09/2010; 107(41):17733-8. · 9.68 Impact Factor
-
Kirk S B Bergstrom,
Vanessa Kissoon-Singh,
Deanna L Gibson,
Caixia Ma, Marinieve Montero,
Ho Pan Sham,
Natasha Ryz,
Tina Huang,
Anna Velcich,
B Brett Finlay,
Kris Chadee,
Bruce A Vallance
[show abstract]
[hide abstract]
ABSTRACT: Despite recent advances in our understanding of the pathogenesis of attaching and effacing (A/E) Escherichia coli infections, the mechanisms by which the host defends against these microbes are unclear. The goal of this study was to determine the role of goblet cell-derived Muc2, the major intestinal secretory mucin and primary component of the mucus layer, in host protection against A/E pathogens. To assess the role of Muc2 during A/E bacterial infections, we inoculated Muc2 deficient (Muc2(-/-)) mice with Citrobacter rodentium, a murine A/E pathogen related to diarrheagenic A/E E. coli. Unlike wildtype (WT) mice, infected Muc2(-/-) mice exhibited rapid weight loss and suffered up to 90% mortality. Stool plating demonstrated 10-100 fold greater C. rodentium burdens in Muc2(-/-) vs. WT mice, most of which were found to be loosely adherent to the colonic mucosa. Histology of Muc2(-/-) mice revealed ulceration in the colon amid focal bacterial microcolonies. Metabolic labeling of secreted mucins in the large intestine demonstrated that mucin secretion was markedly increased in WT mice during infection compared to uninfected controls, suggesting that the host uses increased mucin release to flush pathogens from the mucosal surface. Muc2 also impacted host-commensal interactions during infection, as FISH analysis revealed C. rodentium microcolonies contained numerous commensal microbes, which was not observed in WT mice. Orally administered FITC-Dextran and FISH staining showed significantly worsened intestinal barrier disruption in Muc2(-/-) vs. WT mice, with overt pathogen and commensal translocation into the Muc2(-/-) colonic mucosa. Interestingly, commensal depletion enhanced C. rodentium colonization of Muc2(-/-) mice, although colonic pathology was not significantly altered. In conclusion, Muc2 production is critical for host protection during A/E bacterial infections, by limiting overall pathogen and commensal numbers associated with the colonic mucosal surface. Such actions limit tissue damage and translocation of pathogenic and commensal bacteria across the epithelium.
PLoS Pathogens 05/2010; 6(5):e1000902. · 9.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Peptide "mimics" (mimotopes) of linear protein epitopes and carbohydrate epitopes have been successfully used as immunogens to elicit cross-reactive antibodies against their cognate epitopes; however, immunogenic mimicry has been difficult to achieve for discontinuous protein epitopes. To explore this, we developed from phage-displayed peptide libraries optimized peptide mimics for three well-characterized discontinuous epitopes on hen egg lysozyme and horse cytochrome c. The peptides competed with their cognate antigens for antibody binding, displayed affinities in the nM range, and shared critical binding residues with their native epitopes. Yet, while immunogenic, none of the peptides elicited antibodies that cross-reacted with their cognate antigens. We analyzed the 3-D structure of the site within each discontinuous epitope that shared critical binding residues with its peptide mimic, and observed that in each case it formed a ridge-like patch on the epitope; in no case did it cover most or all of the epitope. Thus, the peptides' lack of immunogenic mimicry could be attributed to their inability to recapitulate the topological features of their cognate epitopes. Our results suggest that direct peptide immunizations are not a practical strategy for generating targeted antibody responses against discontinuous epitopes.
Molecular Immunology 02/2010; 47(5):1137-48. · 2.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Inflammatory bowel diseases (IBD) are thought to occur because of impaired mucosal integrity that allows enteric bacteria to leak out of the intestine, triggering maladaptive intestinal inflammation. While the exact pathogenesis of IBD is unclear, studies have recently demonstrated that bacterial activation of innate receptors not only causes inflammation, but may also play an important role in modulating intestinal epithelial barrier function, as well as intestinal epithelial cell proliferation and repair. These mucosal homeostatic mechanisms are essential in limiting as well as repairing mucosal damage. Therefore bacterial activation of the innate immune system can have both inflammatory as well as protective healing roles in the intestine. Strikingly, these findings suggest that dysregulation of these processes could be responsible for both the barrier dysfunction as well as the heightened inflammatory tone that characterize IBD. In this review we explore the current state of knowledge underlying this novel role for innate immunity in the gastrointestinal tract, and discuss the strengths and weaknesses of the chemical and infectious models used in these studies. In addition, we discuss preliminary evidence that exaggerated microbial activation of the innate immune system may cause the fibrotic responses that develop in some patients with IBD.
Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry (Formerly Cu rrent Medicinal Chemistry - Anti-Inflammatory and Anti-Allergy Agents) 08/2009; 8(3):228-247.
-
[show abstract]
[hide abstract]
ABSTRACT: Enormous efforts have been made to produce a protective vaccine against human immunodeficiency virus type 1; there has been little success. However, the identification of broadly neutralizing antibodies against epitopes on the highly conserved membrane-proximal external region (MPER) of the gp41 envelope protein has delineated this region as an attractive vaccine target. Furthermore, emerging structural information on the MPER has provided vaccine designers with new insights for building relevant immunogens. This review describes the current state of the field regarding (i) the structure and function of the gp41 MPER; (ii) the structure and binding mechanisms of the broadly neutralizing antibodies 2F5, 4E10, and Z13; and (iii) the development of an MPER-targeting vaccine. In addition, emerging approaches to vaccine design are presented.
Microbiology and molecular biology reviews: MMBR 04/2008; 72(1):54-84, table of contents. · 12.59 Impact Factor
-
Erica Ollmann Saphire, Marinieve Montero,
Alfredo Menendez,
Nienke E van Houten,
Melita B Irving,
Ralph Pantophlet,
Michael B Zwick,
Paul W H I Parren,
Dennis R Burton,
Jamie K Scott,
Ian A Wilson
[show abstract]
[hide abstract]
ABSTRACT: The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.
Journal of Molecular Biology 07/2007; 369(3):696-709. · 4.00 Impact Factor
