NCR3/NKp30 contributes to pathogenesis in primary Sjogren's syndrome

Abstract

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by a lymphocytic exocrinopathy. However, patients often have evidence of systemic autoimmunity, and they are at markedly increased risk for the development of non- Hodgkin's lymphoma. Similar to other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, although the precise etiology remains uncertain. NCR3/NKp30 is a natural killer (NK)-specific activating receptor regulating the cross talk between NK and dendritic cells and type II IFN secretion. We performed a case-control study of genetic polymorphisms of the NCR3/NKp30 gene and found that rs11575837 (G>A) residing in the promoter was associated with reduced gene transcription and function as well as protection to pSS. We also demonstrated that circulating levels of NCR3/NKp30 were significantly increased among pSS patients compared with controls and correlated with higher NCR3/NKp30 but not CD16-dependent IFN-γ secretion by NK cells. Excess accumulation of NK cells in minor salivary glands correlated with the severity of the exocrinopathy. B7H6, the ligand of NKp30, was expressed by salivary epithelial cells. These findings suggest that NK cells may promote an NKp30-dependent inflammatory state in salivary glands and that blockade of the B7H6/NKp30 axis could be clinically relevant in pSS.

Figure 1. The rs11575837 minor allele (A) is protective for pSS risk and is associated with reduced NKp30 expression levels

Relative NKp30 mRNA expression in peripheral blood mononuclear cells from patients with pSS, according to genotype: C/G: carriers of rs2736191C minor allele and rs11575837G major allele, G/A: carriers of rs2736191G major allele and rs11575837A minor allele, G/G: carriers of both major alleles. Results represent the mean ± standard error of the mean (SEM). ns: non- significant, * p<0.05

Figure 2. Elevated expression of NKp30 among pSS patients

Phenotypic characterization using flow cytometry analyses of 38 pSS patients and 30 control donors. pSS patients are lymphopenic (A, left) and this lymphopenia is more severe in patients with active disease defined by an ESSDAI ≥ 4 (A, right panel). The proportion of circulating CD3⁻CD56⁺ NK cells (gated on CD45⁺ cells) is significantly reduced in pSS patients versus controls (B) but the proportion of CD56^bright cells (gated on NK cells) increased in patients (C). The NKp30 activating receptor is markedly overexpressed in NK cells [in percentages (D), and in mean fluorescence intensity (E)] in pSS patients. Results represent the mean ± standard error of the mean (SEM). ns: non-significant, * p< 0.05, ** p< 0.001

Figure 3. Functional relevance of a high expression level of NKp30 on NK cells

Proportions of IFN-γ positive NK cells determined using intracellular stainings in flow cytometry analyses after a 24hr- stimulation of PBMC with NKp30 or FcγRIII (CD16) (right panels) antibodies. Linear regression with NKp30 expression levels. A dose response (using 2.5, 0.8 and 0.3 µg/ml of anti -NKp30 Ab coated onto plastic dishes) is depicted, each dot representing one patient or control. Four individuals carrying the minor allele of the rs11575837 (11575837A) were assessed in parallel and the results are indicated in red dots. ns: non-significant, * p< 0.05, ** p< 0.001

Figure 3. Functional relevance of a high expression level of NKp30 on NK cells

Proportions of IFN-γ positive NK cells determined using intracellular stainings in flow cytometry analyses after a 24hr- stimulation of PBMC with NKp30 or FcγRIII (CD16) (right panels) antibodies. Linear regression with NKp30 expression levels. A dose response (using 2.5, 0.8 and 0.3 µg/ml of anti -NKp30 Ab coated onto plastic dishes) is depicted, each dot representing one patient or control. Four individuals carrying the minor allele of the rs11575837 (11575837A) were assessed in parallel and the results are indicated in red dots. ns: non-significant, * p< 0.05, ** p< 0.001

Figure 4. Accumulation of NK cells in minor salivary gland biopsy (MSGB)

A representative NKp46 staining of a paraffin-embedded specimen from pSS and sicca patients. pSS (right micrograph pictures) and sicca controls (left panel): magnification 250X and 945X (inset) (right panel) or 200X (left panel). Scale bars indicated. B-C. Correlation between NK cell numbers and focus score (B) or grade (C) in different areas of the glands. In the Chisholm-Mason scale (54), a grade III (equivalent to 1 focus/4 mm²) or IV (equivalent to >1 focus/4 mm²) are suggestive of the diagnosis of pSS. The numbers of NKp46 positive cells within (right panels) versus outside the foci (left panels) were evaluated in 40 fields observed at x200 power of magnification in 23 MSGB grade GIII and IV and 17 MSGB GI and II. Each dot represents one MSBG. ns: non-significant, * p< 0.05, Mann-Whitney t-test.

Figure 5. TNF-α induces the cross-talk between salivary glands and NK cells in an NKp30-dependent manner

A-B. Transcriptional levels of B7-H6 in salivary glands and regulation. B7-H6 mRNA expression was assessed by quantitative RT-PCR. B7-H6 mRNA expression was assessed from HSG cell line or salivary glandular epithelial cells derived from minor salivary gland biopsy (SGEC), at baseline (biopsy of 8 glands, A) and after a 6 hours-incubation with TNF-α (1 ng/ml), poly I:C (30 µg/ml), IL-17, IL-22 or IL-23 (10ng/mL) (B). C. Functional cross-talk between HSG and NKp30 overexpressing Jurkat cells stimulated by TNF-α. TNF-α -stimulated HSG cells were incubated with Jurkat cells transduced with the three major NKp30 isoforms or with the control vector for 24 hours. The secretion of IL-2 was monitored by ELISA and a histogram of two independent experiments is shown. D. Role of NKp30 ligands/NKp30 interaction in the dialogue between HSG and transduced Jurkat cells. NKp30A–transduced Jurkat cells were incubated with anti-NKp30 or irrelevant blocking antibody for 2 hours prior to stimulation with HSG (as described in B). A representative histogram of two experiments is shown. ** p< 0.001, *** p<0.0001, Two-way Anova.