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Functional perturbation of classical natural killer and innate lymphoid cells in the oral mucosa during SIV infection.

Li H, Reeves RK - Front Immunol (2013)

Bottom Line: Also in contrast to what we have previously found in gut tissues of SIV-infected macaques, we found no reduction in NK cells during chronic SIV infection, but rather an expansion of ILCs in oral-draining lymph nodes and tonsils.These data suggest that the lentivirus-induced depletion of the NK cell/ILC compartment in the gut may be absent in the oral mucosa, but the inherent differences and SIV-induced alterations are likely to have significant impact on preventing oral opportunistic infections in lentiviral disease.Furthermore, these data extend our understanding of the oral innate immune system in general and could aid future studies evaluating the regulation of both normal oral flora and limiting transmission of oral mucosal pathogens.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive Southborough, MA, USA.

ABSTRACT
Despite the fact that the majority of human pathogens are transmitted across mucosal surfaces, including the oral mucosae, oral immunity is poorly understood. Furthermore, because the normal flora of the oral cavity is vast and significantly diverse, host immunity must balance a complex system of tolerance and pathogen recognition. Due to the rapid recognition and response to pathogens, the innate immune system, including natural killer (NK) cells, likely plays a critical role in mediating this balance. Because logistical and ethical restraints limit access to significant quantities of human mucosal tissues, non-human primate models offer one of the best opportunities to study mucosal NK cells. In this study we have identified both classical NK cells, as well as innate lymphoid cells (ILCs) in tonsillar and buccal tissues and oral-draining lymph nodes. Identified by mutually exclusive expression of NKG2A and NKp44, NK cells, and ILCs in the oral mucosa are generally phenotypically and functionally analogous to their gut counterparts. NKG2A(+) NK cells were more cytotoxic while NKp44(+) ILCs produced copious amounts of IL-17 and TNF-α. However, in contrast to gut, oral NK cells and ILCs both produced large quantities of IFN-γ and the beta-chemokine, MIP-1β. Also in contrast to what we have previously found in gut tissues of SIV-infected macaques, we found no reduction in NK cells during chronic SIV infection, but rather an expansion of ILCs in oral-draining lymph nodes and tonsils. These data suggest that the lentivirus-induced depletion of the NK cell/ILC compartment in the gut may be absent in the oral mucosa, but the inherent differences and SIV-induced alterations are likely to have significant impact on preventing oral opportunistic infections in lentiviral disease. Furthermore, these data extend our understanding of the oral innate immune system in general and could aid future studies evaluating the regulation of both normal oral flora and limiting transmission of oral mucosal pathogens.

No MeSH data available.


Related in: MedlinePlus

Oral-associated mucosal tissues contain classical NK cells and innate lymphoid cells. (A) Representative gating strategy to identify NKG2A+ NK cells and NKp44+ ILCs among live mononuclear cells. (B) Representative histograms of NKG2D and NKp30 expression on NK cells and ILCs. Frequencies of NK cells (C) and ILCs (D) among live CD45+CD3− mononuclear cells in tissues of naïve and SIV-infected macaques are shown. Bar and whisker plots represent medians and ranges of 4–12 animals per group. CBx, colorectal biopsy; MLN, mesenteric lymph node; OLN, oral lymph node.
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Figure 1: Oral-associated mucosal tissues contain classical NK cells and innate lymphoid cells. (A) Representative gating strategy to identify NKG2A+ NK cells and NKp44+ ILCs among live mononuclear cells. (B) Representative histograms of NKG2D and NKp30 expression on NK cells and ILCs. Frequencies of NK cells (C) and ILCs (D) among live CD45+CD3− mononuclear cells in tissues of naïve and SIV-infected macaques are shown. Bar and whisker plots represent medians and ranges of 4–12 animals per group. CBx, colorectal biopsy; MLN, mesenteric lymph node; OLN, oral lymph node.

Mentions: To identify NK cell and ILC populations in oral mucosae and draining lymph nodes, we utilized polychromatic flow cytometry employing gating strategies previously developed specifically for rhesus macaques (Reeves et al., 2011). We first gated on CD45+ leukocytes to exclude any contaminating epithelial cells and excluded dead cells using LIVE/DEAD cell stain (Figure 1A). Among live CD45+CD3− mononuclear cells we first identified classical NK cells as NKG2A+—NKG2A has been previously found to be the most useful NK cell inclusive marker for rhesus macaques and other Old World monkeys, despite its variability of expression in humans (Webster and Johnson, 2005; Pereira et al., 2008; Reeves et al., 2010). NKG2A+ NK cells were readily distinguished from ILCs that expressed high levels of NKp44, as previously described for GI tissues (Reeves et al., 2011). NK cells and ILCs also lacked CD20 and CD14 expression (data not shown). To further confirm the identity of oral NK cells, we also analyzed expression of NKG2D and NKp30, which were found at high levels on NKG2A+ NK cells, but were dim to negative on NKp44+ ILCs (Figure 1B). The frequency of NK cells and ILCs also varied widely in different oral tissues. (Figures 1C,D) To provide a frame of reference, we compared NK cells in oral tissues to another mucosal site, colorectum, as well as a distal mucosa-draining lymph node, mesenteric lymph nodes (MLN), and peripheral blood mononuclear cells (PBMC). Classical NKG2A+ NK cells were found at relatively similar frequencies in buccal and colorectal mucosae (median percentage among mononuclear cells, 1.7 and 3.8%, respectively). However, NK cells were ~10-fold less frequent in tonsils and OLN and MLN (median percentage among mononuclear cells, 0.11, 0.29, and 0.44%, respectively). Although in comparison to NK cells, NKp44+ ILCs are rare, they were by far most abundant in colorectal mucosa (median frequency, 1.1%), the tissue where we first described these cells in rhesus macaques. ILCs were detectable in tonsils and lymph nodes, albeit generally lower than 0.05% of mononuclear cells. As expected, NK cells were found at relatively high frequencies in blood, whereas NKp44+ ILCs were virtually absent. NK cells could be further delineated by expression of CD56 and CD16 into four subpopulations and distribution was highly disparate among tissues (Figure 2). As expected, peripheral blood was heavily dominated by CD16+ NK cells, whereas mucosal tissues were enriched for CD56+ NK cells. Although multiple studies have shown this phenomenon, this is the first report that the CD56+ enrichment is also present in buccal mucosa. OLN and tonsils were enriched for CD56+ and DN NK cells similar to distal lymph nodes sites, but generally lacked CD16+ NK cells.


Functional perturbation of classical natural killer and innate lymphoid cells in the oral mucosa during SIV infection.

Li H, Reeves RK - Front Immunol (2013)

Oral-associated mucosal tissues contain classical NK cells and innate lymphoid cells. (A) Representative gating strategy to identify NKG2A+ NK cells and NKp44+ ILCs among live mononuclear cells. (B) Representative histograms of NKG2D and NKp30 expression on NK cells and ILCs. Frequencies of NK cells (C) and ILCs (D) among live CD45+CD3− mononuclear cells in tissues of naïve and SIV-infected macaques are shown. Bar and whisker plots represent medians and ranges of 4–12 animals per group. CBx, colorectal biopsy; MLN, mesenteric lymph node; OLN, oral lymph node.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539714&req=5

Figure 1: Oral-associated mucosal tissues contain classical NK cells and innate lymphoid cells. (A) Representative gating strategy to identify NKG2A+ NK cells and NKp44+ ILCs among live mononuclear cells. (B) Representative histograms of NKG2D and NKp30 expression on NK cells and ILCs. Frequencies of NK cells (C) and ILCs (D) among live CD45+CD3− mononuclear cells in tissues of naïve and SIV-infected macaques are shown. Bar and whisker plots represent medians and ranges of 4–12 animals per group. CBx, colorectal biopsy; MLN, mesenteric lymph node; OLN, oral lymph node.
Mentions: To identify NK cell and ILC populations in oral mucosae and draining lymph nodes, we utilized polychromatic flow cytometry employing gating strategies previously developed specifically for rhesus macaques (Reeves et al., 2011). We first gated on CD45+ leukocytes to exclude any contaminating epithelial cells and excluded dead cells using LIVE/DEAD cell stain (Figure 1A). Among live CD45+CD3− mononuclear cells we first identified classical NK cells as NKG2A+—NKG2A has been previously found to be the most useful NK cell inclusive marker for rhesus macaques and other Old World monkeys, despite its variability of expression in humans (Webster and Johnson, 2005; Pereira et al., 2008; Reeves et al., 2010). NKG2A+ NK cells were readily distinguished from ILCs that expressed high levels of NKp44, as previously described for GI tissues (Reeves et al., 2011). NK cells and ILCs also lacked CD20 and CD14 expression (data not shown). To further confirm the identity of oral NK cells, we also analyzed expression of NKG2D and NKp30, which were found at high levels on NKG2A+ NK cells, but were dim to negative on NKp44+ ILCs (Figure 1B). The frequency of NK cells and ILCs also varied widely in different oral tissues. (Figures 1C,D) To provide a frame of reference, we compared NK cells in oral tissues to another mucosal site, colorectum, as well as a distal mucosa-draining lymph node, mesenteric lymph nodes (MLN), and peripheral blood mononuclear cells (PBMC). Classical NKG2A+ NK cells were found at relatively similar frequencies in buccal and colorectal mucosae (median percentage among mononuclear cells, 1.7 and 3.8%, respectively). However, NK cells were ~10-fold less frequent in tonsils and OLN and MLN (median percentage among mononuclear cells, 0.11, 0.29, and 0.44%, respectively). Although in comparison to NK cells, NKp44+ ILCs are rare, they were by far most abundant in colorectal mucosa (median frequency, 1.1%), the tissue where we first described these cells in rhesus macaques. ILCs were detectable in tonsils and lymph nodes, albeit generally lower than 0.05% of mononuclear cells. As expected, NK cells were found at relatively high frequencies in blood, whereas NKp44+ ILCs were virtually absent. NK cells could be further delineated by expression of CD56 and CD16 into four subpopulations and distribution was highly disparate among tissues (Figure 2). As expected, peripheral blood was heavily dominated by CD16+ NK cells, whereas mucosal tissues were enriched for CD56+ NK cells. Although multiple studies have shown this phenomenon, this is the first report that the CD56+ enrichment is also present in buccal mucosa. OLN and tonsils were enriched for CD56+ and DN NK cells similar to distal lymph nodes sites, but generally lacked CD16+ NK cells.

Bottom Line: Also in contrast to what we have previously found in gut tissues of SIV-infected macaques, we found no reduction in NK cells during chronic SIV infection, but rather an expansion of ILCs in oral-draining lymph nodes and tonsils.These data suggest that the lentivirus-induced depletion of the NK cell/ILC compartment in the gut may be absent in the oral mucosa, but the inherent differences and SIV-induced alterations are likely to have significant impact on preventing oral opportunistic infections in lentiviral disease.Furthermore, these data extend our understanding of the oral innate immune system in general and could aid future studies evaluating the regulation of both normal oral flora and limiting transmission of oral mucosal pathogens.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive Southborough, MA, USA.

ABSTRACT
Despite the fact that the majority of human pathogens are transmitted across mucosal surfaces, including the oral mucosae, oral immunity is poorly understood. Furthermore, because the normal flora of the oral cavity is vast and significantly diverse, host immunity must balance a complex system of tolerance and pathogen recognition. Due to the rapid recognition and response to pathogens, the innate immune system, including natural killer (NK) cells, likely plays a critical role in mediating this balance. Because logistical and ethical restraints limit access to significant quantities of human mucosal tissues, non-human primate models offer one of the best opportunities to study mucosal NK cells. In this study we have identified both classical NK cells, as well as innate lymphoid cells (ILCs) in tonsillar and buccal tissues and oral-draining lymph nodes. Identified by mutually exclusive expression of NKG2A and NKp44, NK cells, and ILCs in the oral mucosa are generally phenotypically and functionally analogous to their gut counterparts. NKG2A(+) NK cells were more cytotoxic while NKp44(+) ILCs produced copious amounts of IL-17 and TNF-α. However, in contrast to gut, oral NK cells and ILCs both produced large quantities of IFN-γ and the beta-chemokine, MIP-1β. Also in contrast to what we have previously found in gut tissues of SIV-infected macaques, we found no reduction in NK cells during chronic SIV infection, but rather an expansion of ILCs in oral-draining lymph nodes and tonsils. These data suggest that the lentivirus-induced depletion of the NK cell/ILC compartment in the gut may be absent in the oral mucosa, but the inherent differences and SIV-induced alterations are likely to have significant impact on preventing oral opportunistic infections in lentiviral disease. Furthermore, these data extend our understanding of the oral innate immune system in general and could aid future studies evaluating the regulation of both normal oral flora and limiting transmission of oral mucosal pathogens.

No MeSH data available.


Related in: MedlinePlus