ILC1s and ILC3s Exhibit Inflammatory Phenotype in Periodontal Ligament of Periodontitis Patients.

Innate lymphoid cells (ILCs) are emerging as important players in inflammatory diseases. The oral mucosal barrier harbors all ILC subsets, but how these cells regulate the immune responses in periodontal ligament tissue during periodontitis remains undefined. Here, we show that total ILCs are markedly increased in periodontal ligament of periodontitis patients compared with healthy controls. Among them, ILC1s and ILC3s, particularly NKp44+ILC3 subset, are the predominant subsets accumulated in the periodontal ligament. Remarkably, ILC1s and ILC3s from periodontitis patients produce more IL-17A and IFN-γ than that from healthy controls. Collectively, our results highlight the role of ILCs in regulating oral immunity and periodontal ligament inflammation and provide insights into targeting ILCs for the treatment of periodontitis.

Differential Effect of Mucosal NKp44+ Innate Lymphoid Cells and Δγ Cells on Simian Immunodeficiency Virus Infection Outcome in Rhesus Macaques.

NK cells are essential for controlling viral infections. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following mucosal priming with replicating adenovirus (Ad)-SIV recombinants, systemic boosting with SIV envelope protein, and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. No differences in NK cell or ILC frequencies or cytokine production were seen between vaccinated and Ad-empty/alum controls, suggesting responses were due to the Ad-vector and alum vaccine components. Mucosal NKp44+ ILCs increased postvaccination and returned to prelevels postinfection. The vaccine regimen induced mucosal SIV-specific Ab, which mediated Ab-dependent cellular cytotoxicity and was correlated with mucosal NKp44+CD16+ ILCs. Postvaccination NKp44+ and NKp44+IL-17+ ILC frequencies were associated with delayed SIV acquisition and decreased viremia. In chronically SIV-infected animals, NKp44+ ILCs negatively correlated with viral load, further suggesting a protective effect, whereas, NKG2A- NKp44- double-negative ILCs positively correlated with viral load, indicating a pathogenic role. No such associations of circulating NK cells were seen. Δγ NK cells in mucosal tissues of chronically infected animals exhibited impaired cytokine production compared with non-Δγ NK cells but responded to anti-gp120 Ab and Gag peptides, whereas non-Δγ NK cells did not. Mucosal Δγ NKp44+ and Δγ DN cells were similarly associated with protection and disease progression, respectively. Thus, the data suggest NKp44+ ILCs and Δγ cells contribute to SIV infection outcomes. Vaccines that promote mucosal NKp44+ and suppress double-negative ILCs are likely desirable.

Profile of natural killer cells after a previous natural Vaccinia virus infection in an in vitro viral re-exposure.

The present study compares the profile of NK cells in an in vitro re-exposure by Vaccinia virus (VACV), in groups that have had a previous vaccination or natural infection. Our data suggests that stimulation with VACV triggers a cytotoxic response by NK cells marked by an increase of NCRs: NKp30, NKp44, and NKp46 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. However, the degranulation and secretion processes are inhibited in infected (vaccinated and unvaccinated) subjects and in the non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. We demonstrated that stimulation with VACV downregulates the percentage of expression of Perforin, Granzyme A, and CD107a, but upregulate CD94 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. Furthermore, the percentage of IFN-γ(+) NK cells was significantly lower in non-infected unvaccinated subjects, when compared with infected (vaccinated and unvaccinated) and non-infected vaccinated individuals. Our results also show that the percentage of TNF-α(+) NK cells was significantly higher in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals, after in vitro stimulation with UV-inactivated VACV. Our data suggest that the expression of NCRs NKp30, NKp44, NKp46 and cytokines by NK cells are important in the innate response against VACV.

Augmenting the expression of NKp44 molecule and the natural killer activity in peripheral blood mononuclear cells from patients with malignant colorectal carcinoma.

OBJECTIVE: NKp44 and NKG2D are of the main NK activating receptors involved in recognition and killing of tumors. Here we studied the stimulatory effects of PHA and/or K562 cell line on induction of NKp44 and NKG2D expression and the NK activity of PBMCs from patients with colorectal carcinoma (CRC). MATERIALS AND METHODS: Peripheral blood samples were collected from 10 patients with CRC. The peripheral blood mononuclear cells (PBMCs) from each patient received a single stimulation with PHA or double stimulation with PHA and irradiated K562 cell line (iK562). The expression of CD56, NKG2D and NKp44 were detected by flowcytometry. The NK activity of PBMCs against a colorectal carcinoma cell line named as SW742 was determined with 51Cr-release assay. RESULTS: Double stimulation of PBMCs with PHA+iK562 significantly augmented the number CD56(+) cells compared to PHA alone and non-stimulated PBMCs (P<0.000, P<0.0000; respectively). A single stimulation of PBMCs with PHA resulted in an enhancement in NKG2D and NKp44 expression from 16.6±3.3% (for non-stimulated PBMCs) to 42±5.6% and 48.1±3.8% respectively (p<0.05). Double stimulation of PBMCs augmented the NKp44 expression significantly in comparison with single stimulation with PHA (73.6±12%, p<0.05). Double stimulation of PBMCs significantly enhanced the NK activity against SW742 target cells compared to single stimulation with PHA (p<0.05). DISCUSSION AND CONCLUSION: Our results demonstrated that the mitogen and iK562 exposure to PBMCs can significantly improve NK activity which is co-related to the higher expression of NKp44 and NKG2D. These data may help to improve cancer immunotherapy protocols.

NKp44L expression on CD4+ T cells is associated with impaired immunological recovery in HIV-infected patients under highly active antiretroviral therapy.

OBJECTIVE: HIV-infected immunological nonresponders (InRs) patients fail to show satisfactory CD4+ T-cell recovery despite virologically effective HAART. We propose that NKp44L, the cellular ligand of an activating natural killer (NK) receptor, expressed only on uninfected bystander CD4+ T cells from HIV-1 infected patients, could play a major role in this phenomenon by sensitizing these cells to NK killing. DESIGN: Phenotype and multifunctional status of CD4+ T cells, especially the subsets expressing and not expressing NKp44L, were characterized for HIV-infected patients receiving HAART for at least 2 years, during which their viral load remained less than 40 copies/ml; 53 were InRs (CD4 cell count always <350 cells/µl), and 82 immunological responders (CD4 cell count always ≥350 cells/µl). Flow cytometry determined NKp44L expression in association with specific markers of proliferation, maturation, activation, homeostasis, and intracellular cytokine production. Degranulation of NKp44+ determined the functional capacity of NK cells. RESULTS: InRs exhibited high levels of NKp44L+CD4+ T cells. Compared with NKp44L negative cells, the frequency of naive CD45RA+CCR7+ T cells expressing NKp44L fell (P < 0.001) and their proliferative capacity grew. Moreover, apoptosis and a unique ability to produce multiple cytokines (IL-2, IFN-γ, and TNF-α) without or after phytohemagglutinin or anti-CD3/CD28 stimulation distinguished NKp44L+ T cells. CONCLUSION: InR status is associated to a significant expansion of highly differentiated, multifunctional and apoptotic CD4+ T cells expressing NKp44L. This could explain a rapid CD4+ T-cell turnover in InR preventing immune recovery. These data suggest a new target for developing therapeutic strategies to prevent NKp44L expression and then stimulating immune recovery in InRs.

Flow cytometry in detection of abnormalities of natural killer cell.

The population of natural killer (NK) cells is very heterogeneous and plays a role in the immune system. Several NK cells subpopulations are recognized, differing in phenotype, cytokine release and cytotoxic ability. Different expression of biologically relevant molecules on the surface of NK cells may indicate their multiple functions. The activity of NK cells has mainly to do with their cytotoxic nature. A complete analysis of NK cells function requires application of many tests because a defect may be present at different stages of the cytotoxic process, from signal transduction through lysosome degranulation to target cells destruction. Flow cytometry is actually one of the best methods for the identification of NK cells and tracking their defects.

Interleukin-22 and interleukin-22-producing NKp44+ natural killer cells in subclinical gut inflammation in ankylosing spondylitis.

OBJECTIVE: The intestinal inflammation observed in patients with ankylosing spondylitis (AS) is characterized by an overexpression of interleukin-23 (IL-23). IL-23 is known to regulate IL-22 production through lamina propria NKp44+ natural killer (NK) cells, which are thought to be involved in protective mucosal mechanisms. This study was undertaken to evaluate the frequency of NKp44+ NK cells and the expression of IL-22 in the ileum of AS patients. METHODS: Tissue NKp44+ NK cells, NKp46+ NK cells, and IL-22-producing cells were analyzed by flow cytometry. Quantitative gene expression analysis of IL-22, IL-23, IL-17, STAT-3, and mucin 1 (MUC-1) was performed by reverse transcriptase-polymerase chain reaction on ileal samples from 15 patients with AS, 15 patients with Crohn's disease (CD), and 15 healthy controls. NKp44, pSTAT-3, and IL-22 expression was analyzed by immunohistochemistry. RESULTS: The frequency of NKp44+ but not NKp46+ NK cells was increased in the inflamed ileum of AS patients compared to CD patients and controls. The frequency of NKp46+ NK cells was significantly increased only in CD patients. Among CD4+ lymphocytes and NKp44+ NK cell subsets, the latter were the major source of IL-22 on lamina propria mononuclear cells from AS patients. Significant up-regulation of IL-22, IL-23p19, MUC-1, and STAT-3 transcripts in the terminal ileum of patients with AS was observed. Immunohistochemical analysis confirmed the increased IL-22 and pSTAT-3 expression in inflamed mucosa from AS and CD patients. CONCLUSION: Our findings indicate that overexpression of IL-22, together with an increased number of IL-22-producing NKp44+ NK cells, occurs in the gut of AS patients, where it appears to play a tissue-protective role.