MHC-II presentation by oral Langerhans cells impacts intraepithelial Tc17 abundance and Candida albicans oral infection via CD4 T cells.

In a murine model (LCΔMHC-II) designed to abolish MHC-II expression in Langerhans cells (LCs), ∼18% of oral LCs retain MHC-II, yet oral mucosal CD4 T cells numbers are unaffected. In LCΔMHC-II mice, we now show that oral intraepithelial conventional CD8αß T cell numbers expand 30-fold. Antibody-mediated ablation of CD4 T cells in wild-type mice also resulted in CD8αß T cell expansion in the oral mucosa. Therefore, we hypothesize that MHC class II molecules uniquely expressed on Langerhans cells mediate the suppression of intraepithelial resident-memory CD8 T cell numbers via a CD4 T cell-dependent mechanism. The expanded oral CD8 T cells co-expressed CD69 and CD103 and the majority produced IL-17A [CD8 T cytotoxic (Tc)17 cells] with a minority expressing IFN-γ (Tc1 cells). These oral CD8 T cells showed broad T cell receptor Vß gene usage indicating responsiveness to diverse oral antigens. Generally supporting Tc17 cells, transforming growth factor-ß1 (TGF-ß1) increased 4-fold in the oral mucosa. Surprisingly, blocking TGF-ß1 signaling with the TGF-R1 kinase inhibitor, LY364947, did not reduce Tc17 or Tc1 numbers. Nonetheless, LY364947 increased γδ T cell numbers and decreased CD49a expression on Tc1 cells. Although IL-17A-expressing γδ T cells were reduced by 30%, LCΔMHC-II mice displayed greater resistance to Candida albicans in early stages of oral infection. These findings suggest that modulating MHC-II expression in oral LC may be an effective strategy against fungal infections at mucosal surfaces counteracted by IL-17A-dependent mechanisms.

Major Histocompatibility Complex II Expression on Oral Langerhans Cells Differentially Regulates Mucosal CD4 and CD8 T Cells.

In murine periodontitis, the T helper (Th)17 response against Porphyromonas gingivalis in cervical lymph node is abrogated by diphtheria toxin-driven depletion of Langerhans cells (LCs). We determined the impact of major histocompatibility complex class II (MHC-II) presentation in LCs on Th17 cells in the oral mucosa of mice. Using an established human-Langerin promoter-Cre mouse model, we generated LC-specific deletion of the H2-Ab1 (MHC-II) gene. MHC-II expression was ablated in 81.2% of oral-resident LCs compared with >99% of skin-resident LCs. MHC-II (LCΔMHC-II) depletion did not reduce the number of CD4 T cells nor the frequency of Th17 cells compared with that in wild-type mice. However, the frequencies of Th1 cells decreased, and Helios+ T-regulatory cells increased. In ligature-induced periodontitis, the numbers of CD4 T cells and Th17 cells were similar in LCΔMHC-II and wild-type mice. Normal numbers of Th17 cells can therefore be sustained by as little as 18.8% of MHC-II-expressing LCs in oral mucosa. Unexpectedly, oral mucosa CD8 T cells increased >25-fold in LCΔMHC-II mice. Hence, these residual MHC-II-expressing LCs appear unable to suppress the local expansion of CD8 T cells while sufficient to sustain a homeostatic CD4 T-cell response. Reducing the expression of MHC-II on specific LC subpopulations may ultimately boost CD8-mediated intraepithelial surveillance at mucosal surfaces.

Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis.

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9-/- and wild-type (WT; S100A9+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.

Transient Expression of IL-17A in Foxp3 Fate-Tracked Cells in Porphyromonas gingivalis-Mediated Oral Dysbiosis.

In periodontitis Porphyromonas gingivalis contributes to the development of a dysbiotic oral microbiome. This altered ecosystem elicits a diverse innate and adaptive immune response that simultaneously involves Th1, Th17, and Treg cells. It has been shown that Th17 cells can alter their gene expression to produce interferon-gamma (IFN-γ). Forkhead box P3 (Foxp3) is considered the master regulator of Treg cells that produce inhibitory cytokines like IL-10. Differentiation pathways that lead to Th17 and Treg cells from naïve progenitors are considered antagonistic. However, it has been reported that Treg cells expressing IL-17A as well as IFN-γ producing Th17 cells have been observed in several inflammatory conditions. Each scenario appears plausible with T cell transdifferentiation resulting from persistent microbial challenge and consequent inflammation. We established that oral colonization with P. gingivalis drives an initial IL-17A dominated Th17 response in the oral mucosa that is dependent on intraepithelial Langerhans cells (LCs). We hypothesized that Treg cells contribute to this initial IL-17A response through transient expression of IL-17A and that persistent mucosal colonization with P. gingivalis drives Th17 cells toward an IFN-γ phenotype at later stages of infection. We utilized fate-tracking mice where IL-17A- or Foxp3-promoter activity drives the permanent expression of red fluorescent protein tdTomato to test our hypothesis. At day 28 of infection timeline, Th17 cells dominated in the oral mucosa, outnumbering Th1 cells by 3:1. By day 48 this dominance was inverted with Th1 cells outnumbering Th17 cells by nearly 2:1. Tracking tdTomato+ Th17 cells revealed only sporadic transdifferentiation to an IFN-γ-producing phenotype by day 48; the appearance of Th1 cells at day 48 was due to a late de novo Th1 response. tdTomato+ Foxp3+ T cells were 35% of the total live CD4+T cells in the oral mucosa and 3.9% of them developed a transient IL-17A-producing phenotype by day 28. Interestingly, by day 48 these IL-17A-producing Foxp3+ T cells had disappeared. Therefore, persistent oral P. gingivalis infection stimulates an initial IL-17A-biased response led by Th17 cells and a small but significant number of IL-17A-expressing Treg cells that changes into a late de novo Th1 response with only sporadic transdifferentiation of Th17 cells.

Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter.

Mouse models that combine specific loxP-flanked gene sequences with Cre recombinase expressed from cell-regulated promoters have become important tools to investigate gene function. Critically however, expression of Cre recombinase may not always be restricted to the target cell or tissue of interest due to promiscuous activity of the driving promoter. Expression of Cre recombinase and, by extension, excision of the loxP-flanked gene may occur in non-target cells and may not be readily apparent. Here we report on the fidelity of Cre recombinase expressed from the il17a or Foxp3 promoters by combining them with a constitutively expressed floxed-stopped tdTomato reporter gene. Foxp3-driven Cre recombinase in F1 mice induced tdTomato red fluorescent protein in Treg cells but also in a range of other immune cells. Frequency of tdTomato expression was variable but positively correlated (p < 0.0001) amongst lymphoid (B cells and CD8 T cells) and blood-resident myeloid cells (dendritic cells, monocytes, neutrophils) suggesting stochastic activity of the Foxp3 promoter rather than developmental regulation in common ancestral progenitors. Interestingly, frequency of tdTomato+ dendritic cells, monocytes and neutrophils did not correlate with the tdTomato+ fraction in eosinophils, indicating that activity of the Foxp3 promoter in eosinophils occurred after the split from a common multipotent progenitor. When these F1 mice were crossed to achieve homozygosity of the promoter and reporter gene, a novel visually red phenotype was observed segregating amongst littermates. The red coloration was widespread and prevalent in non-immune tissues. Thymocytes examined from these red mice showed that all four subsets of immature thymocytes (CD4- CD8-) based on differential expression of CD25 and CD44 were expressing tdTomato. Finally, we show evidence of Foxp3 Cre recombinase independent tdTomato expression, suggesting germ line transmission of an activated tdTomato reporter gene. Our data highlights potential issues with conclusions drawn from using specifically the B6.129(Cg)-Foxp3tm4(YFP/Cre)Ayr/J mice.

Fetal Weight Outcomes in C57BL/6J and C57BL/6NCrl Mice after Oral Colonization with Porphyromonas gingivalis.

Porphyromonas gingivalis is considered a keystone pathogen that contributes to the initiation and progression of periodontitis in humans. P. gingivalis has also been detected in human placentas associated with adverse pregnancy outcomes. The spread of P. gingivalis from the oral cavity to the reproductive tract thus represents a potential mechanism whereby periodontitis can lead to adverse pregnancy outcomes. In a murine model of pregnancy and oral infection with P. gingivalis, C57BL/6J mice developed low fetal weight, whereas C57BL/6NCrl mice did not. Although C57BL/6NCrl mice harbor segmented filamentous bacteria that drive a Th17 response, fetal weight was independent of frequency of Th17 or Th1 in either substrain. Low fetal weight was instead correlated with increasing amounts of P. gingivalis DNA in the placentas of the C57BL/6J dams. In contrast, fetal weight in C57BL/6NCrl mice was independent of P. gingivalis in the placenta. Differences in genetics or microbiome that influence the ability of P. gingivalis to colonize the placenta may drive differential fetal weight outcomes between C57BL/6J and C57BL/6NCrl mice and, potentially, between diverse human populations.

Discriminating between Interstitial and Circulating Leukocytes in Tissues of the Murine Oral Mucosa Avoiding Nasal-Associated Lymphoid Tissue Contamination.

Periodontitis is a chronic inflammatory response to a microbial biofilm that destroys bone and soft tissues supporting the teeth. Murine models of periodontitis based on Porphyromonas gingivalis (Pg) colonization have shown that extravasation of leukocytes into oral tissue is critical to driving alveolar bone destruction. Identifying interstitial leukocytes is key to understanding the immunopathogenesis of periodontitis. Here, we describe a robust flow cytometry assay based on intravenous FITC-conjugated anti-mouse CD45 mAb that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. Unaccounted circulating leukocytes skewed the relative frequency of B cells and granulocytes and inflated the numbers of all leukocyte cell types. We also describe a dissection technique that avoids contamination of oral mucosal tissues with nasal-associated lymphoid tissues (NALT), a B cell rich organ that can inflate leukocyte numbers at least 10-fold and skew the assessment of interstitial CD4 T cell phenotypes. Unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44hi). We report for the first time the presence of antigen-experienced Pg-specific CD4 T cells in NALT following oral feeding of mice with Pg. This new combined flow cytometry and dissection approach allows identification of leukocytes infiltrating the connective tissues of the murine oral mucosa and avoids confounding analyses of leukocytes not recruited to inflamed oral mucosal tissues in disease conditions like periodontitis, candidiasis, or sialadenitis.

Mucosal Langerhans Cells Promote Differentiation of Th17 Cells in a Murine Model of Periodontitis but Are Not Required for Porphyromonas gingivalis-Driven Alveolar Bone Destruction.

Periodontitis is a chronic oral inflammatory disease affecting one in five individuals that can lead to tooth loss. CD4(+) Th cells activated by a microbial biofilm are thought to contribute to the destruction of alveolar bone surrounding teeth by influencing osteoclastogenesis through IL-17A and receptor activator for NF-κB ligand effects. The relative roles of mucosal Ag presentation cells in directing Th cell immune responses against oral pathogens and their contribution to destruction of alveolar bone remain unknown. We tested the contribution of mucosal Langerhans cells (LCs) to alveolar bone homeostasis in mice following oral colonization with a well-characterized human periodontal pathogen, Porphyromonas gingivalis We found that oral mucosal LCs did not protect from or exacerbate crestal alveolar bone destruction but were responsible for promoting differentiation of Th17 cells specific to P. gingivalis. In mice lacking LCs the Th17 response was suppressed and a Th1 response predominated. Bypassing LCs with systemic immunization of P. gingivalis resulted in a predominantly P. gingivalis-specific Th1 response regardless of whether LCs were present. Interestingly, we find that in vivo clonal expansion of P. gingivalis-specific Th cells and induced regulatory T cells does not depend on mucosal LCs. Furthermore, destruction of crestal alveolar bone induced by P. gingivalis colonization occurred regardless of the presence of mucosal LCs or P. gingivalis-specific Th17 cells. Our data indicate that both LCs and Th17 cells are redundant in contributing to alveolar bone destruction in a murine model of periodontitis.

Maintenance of genetic variation in plants and pathogens involves complex networks of gene-for-gene interactions.

The RPP13 [recognition of Hyaloperonospora arabidopsidis (previously known as Peronospora parasitica)] resistance (R) gene in Arabidopsis thaliana exhibits the highest reported level of sequence diversity among known R genes. Consistent with a co-evolutionary model, the matching effector protein ATR13 (A. thaliana-recognized) from H. arabidopsidis reveals extreme levels of allelic diversity. We isolated 23 new RPP13 sequences from a UK metapopulation, giving a total of 47 when combined with previous studies. We used these in functional studies of the A. thaliana accessions for their resistance response to 16 isolates of H. arabidopsidis. We characterized the molecular basis of recognition by the expression of the corresponding ATR13 genes from these 16 isolates in these host accessions. This allowed the determination of which alleles of RPP13 were responsible for pathogen recognition and whether recognition was dependent on the RPP13/ATR13 combination. Linking our functional studies with phylogenetic analysis, we determined that: (i) the recognition of ATR13 is mediated by alleles in just a single RPP13 clade; (ii) RPP13 alleles in other clades have evolved the ability to detect other pathogen ATR protein(s); and (iii) at least one gene, unlinked to RPP13 in A. thaliana, detects a different subgroup of ATR13 alleles.

Host-parasite coevolutionary conflict between Arabidopsis and downy mildew.

Plants are constantly exposed to attack by an array of diverse pathogens but lack a somatically adaptive immune system. In spite of this, natural plant populations do not often suffer destructive disease epidemics. Elucidating how allelic diversity within plant genes that function to detect pathogens (resistance genes) counteracts changing structures of pathogen genes required for host invasion (pathogenicity effectors) is critical to our understanding of the dynamics of natural plant populations. The RPP13 resistance gene is the most polymorphic gene analyzed to date in the model plant Arabidopsis thaliana. Here we report the cloning of the avirulence gene, ATR13, that triggers RPP13-mediated resistance, and we show that it too exhibits extreme levels of amino acid polymorphism. Evidence of diversifying selection visible in both components suggests that the host and pathogen may be locked in a coevolutionary conflict at these loci, where attempts to evade host resistance by the pathogen are matched by the development of new detection capabilities by the host.

The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana.

We have used the naturally occurring plant-parasite system of Arabidopsis thaliana and its common parasite Peronospora parasitica (downy mildew) to study the evolution of resistance specificity in the host population. DNA sequence of the resistance gene, RPP13, from 24 accessions, including 20 from the United Kingdom, revealed amino acid sequence diversity higher than that of any protein coding gene reported so far in A. thaliana. A significant excess of amino acid polymorphism segregating within this species is localized within the leucine-rich repeat (LRR) domain of RPP13. These results indicate that single alleles of the gene have not swept through the population, but instead, a diverse collection of alleles have been maintained. Transgenic complementation experiments demonstrate functional differences among alleles in their resistance to various pathogen isolates, suggesting that the extreme amino acid polymorphism in RPP13 is maintained through continual reciprocal selection between host and pathogen.

Use of suppression subtractive hybridization to identify downy mildew genes expressed during infection of Arabidopsis thaliana.

SUMMARY Peronospora parasitica is an obligate biotrophic oomycete that causes downy mildew in Arabidopsis thaliana and Brassica species. Our goal is to identify P. parasitica (At) genes that are involved in pathogenicity. We used suppression subtractive hybridization (SSH) to generate cDNA libraries enriched for in planta-expressed parasite genes and up-regulated host genes. A total of 1345 clones were sequenced representing cDNA fragments from 25 putative P. parasitica (At) genes (Ppat 1-25) and 618 Arabidopsis genes. Analyses of expression patterns showed that 15 Ppats were expressed only in planta. Eleven Ppats encoded peptides with homology (BlastP values < 1e-05) to proteins with roles in membrane or cell wall biosynthesis, amino acid metabolism, osmoregulation, cation transport, phosphorylation or protein secretion. The other 14 represent potentially novel oomycete genes with none having homologues in an extensive Phytophthora species EST database. A full-length sequence was obtained for four Ppats and each encoded small cysteine-rich proteins with amino-terminal signal peptide sequences. These results demonstrate the utility of SSH in obtaining novel in planta-expressed genes from P. parasitica (At) that complements other gene discovery approaches such as EST sequencing.