Cytokine Production after TLR 2 Stimulation
There were no significant differences in the number of CD14+ monocytes isolated from children with ASD (0.33 ± 0.04 × 106 cells/ml; mean ± S.E.M.) compared with TD controls (0.36 ± 0.03 × 106 cells/ml, p = ns). In addition, there were no differences in the frequencies of putative monocyte subsets CD14+CD16+ and CD14+CD16- cells in children with ASD compared with TD controls (data not shown). To determine the effects of specific TLR2 ligand stimulation, monocyte cell cultures were stimulated with LTA for 24 hours, after which the supernatants were analyzed for cytokine and chemokine production by Luminex multiplex analysis. Monocyte cell cultures from children with ASD were found to produce significantly increased levels of IL-1β (652.8 ± 197.6 pg/ml; mean ± S.E.M.) compared with TD controls (178.4 ± 29.4 pg/ml, p = 0.02, ). IL-6 production was also significantly increased from monocyte cell cultures from children with ASD that had been stimulated with LTA (244,300 ± 80,710 pg/ml) compared with TD controls (78,270 ± 26,100 pg/ml, p = 0.04, ). Similarly, TNFα production was significantly elevated in LTA-stimulated monocyte cell cultures from children with ASD (5,795 ± 1,786 pg/ml) compared with TD controls (2,004 ± 1,826 pg/ml, p = 0.04, ). LTA-induced production of IL-8 (477,578 ± 390,757 ASD vs. 85,982 ± 18,178 TD pg/ml), and GM-CSF (51.9 ± 8.1 ASD vs. 42.9 ± 5.0 TD pg/ml) followed a trend towards increased cytokine production in ASD but did not reach statistical significance between ASD and TD controls. The concentration of MCP-1 following LTA-stimulation was not different between ASD and TD (20,815 ± 5,405 ASD vs. 39,952 ± 10,163 TD pg/ml).
Figure 1 Induced cytokine concentrations of IL-1β (A, D), IL-6 (B, E), and TNFα (C. F) in monocyte cell culture supernatants following stimulation with the TLR 2 ligand LTA (A-C) or TLR4 ligand LPS (D-F). Responses are shown for supernatants from (more ...)
Flow Cytometry Analysis of TLR 2 and HLA-DR Cell Surface Expression
To determine the effects of TLR 2 ligand stimulation on the cell surface expression of its receptor and the activation marker HLA-DR, CD14+ monocytes were evaluated for cell surface expression of, TLR 2 and HLA-DR by flow cytometry after a 24-hour culture in the presence of media alone or the TLR 2 ligand LTA as described above. The frequency of cells that showed positive staining for TLR 2 () were not significantly different in children with ASD compared with TD controls after incubation with media or with the TLR 2 ligand LTA. Mean fluorescent intensity (MFI) of TLR 2 was not significantly different between children with ASD compared with TD controls following stimulation with media or after TLR 2 ligand stimulation (). As anticipated following 24 hour culture, the majority of CD14+ monocytes showed positive staining for HLA-DR from children with ASD and TD controls (). There was an increased frequency of monocyts that had positive staining for HLA-DR in children with ASD compared to TD controls after incubation in media alone (Mean + SEM, ASD vs. TD, 78.48 ± 3.43 vs. 68.06 ± 4.98, p = 0.01, ) and following TLR 2 stimulation (73.5 ± 4.5 vs. 61.19 ± 4.96, p = 0.02, ). The MFI of HLA-DR expression did not differ significantly in children with ASD compared with TD controls ().
Figure 2 Frequency (%) of CD14+ monocytes that demonstrate positive TLR-2 staining in children with ASD (white bars) or TD controls (grey bars) following 24-hour incubation with media alone or LTA (A). Mean fluorescent intensity (MFI) of TLR-2 staining on CD14+ (more ...)
Figure 3 The frequency (%) of CD14+ monocytes that demonstrate positive staining for HLA-DR in children with ASD (white bars) or TD controls (grey bars) following 24-hour incubation with media alone or LTA (A). The frequency of HLA-DR+ CD14+ monocytes is increased (more ...)
Cytokine Production after TLR 4 Stimulation
IL-1β, IL-6 and TNFα concentrations following monocyte cell culture stimulation with the TLR 4 ligand LPS as measured by Luminex are shown in D-F. Monocyte cell cultures from children with ASD responded to LPS stimulation by producing significantly increased concentration of IL-1β when compared with TD controls (2,418 ± 417 pg/ml vs. 1,312 ± 267 pg/ml respectively, p = 0.04, ). However, there were no significant differences in IL-6 (210,100 ± 64,250 pg/ml vs. 179,600 ± 49,440 pg/ml, ASD vs TD, ) or TNFα production (5,249 ± 1,126 pg/ml vs. 6,235 ± 1,079 pg/ml, ) from LPS-stimulated monocyte cell cultures of children with ASD compared with TD controls. Similarly, there were no differences in the production of IL-8 (113,741 ± 20,120 vs. 82,110 ±. 7,679 pg/ml) or GM-CSF (88.7 ± 12.9 vs. 116.9 ± 18.3 pg/ml) between ASD and TD following LPS stimulation. However, the concentration of the chemokine MCP-1 was significantly decreased in monocyte cell cultures from ASD children compared with TD controls after LPS stimulation (2,864 ± 535 pg/ml vs. 25,570 ± 7,123 pg/ml, p = 0.005). We then examined whether there were associations between induced cytokine levels following TLR 4 stimulation and clinical variables among ASD participants. The release of IL-1β following LPS stimulation was associated with impairments in social interactions (rho = 0.56, p = 0.039) and non-verbal communication (rho = 0.87, p = 0.006) as measured using the ADI-R. Similarly, the release of IL-6 following LPS stimulation was associated with non-verbal communication (rho = 0.84, p = 0.009). An association between IL-6 release following LPS stimulation with social interactions (rho = 0.50, p = 0.067) as measured by ADI-R did not reach statistical significance but there was a statistically significant association with social interactions (rho = 0.81, p = 0.05) as measured by the ADOS. These data suggest that more impaired social behaviors and nonverbal communication are associated with increased release of IL-1β and IL-6 after LPS stimulation. No associations were observed between behavioral assessments and with other cytokines or for induced cytokine responses following stimulation with any other TLR ligands investigated. There were no associations between induced cytokine levels and gastrointestinal symptoms.
Cytokine Production after TLR 3, TLR 5, and TLR 9 stimulation
As children with ASD had demonstrated increased sensitivity to signaling via TLR 2 and TLR 4 with resultant cytokine production of pro-inflammatory cytokines, we hypothesized that children with ASD may have a global alteration in TLR signaling to all PAMPs. To determine if monocyte cell culture production of proinflammatory IL-1β, IL-6, and TNFα was elevated for other TLR ligands, we assessed induced cytokine production by Luminex following stimulation with either flagellin (TLR 5), poly I:C (TLR 3) or CpG-B (TLR 9). In contrast to stimulation with either LTA or LPS, monocyte cell cultures from children with ASD did not produced significantly different levels in the majority of cytokines that were assessed, after stimulation with either poly I:C or flagellin when compared with monocytes cell cultures from TD controls that were similarly stimulated (). In contrast, MCP-1 was decreased in ASD children compared to TD controls following poly (I:C) stimulation (28,630 ± 4,952 vs. 52,500 ± 6,178 pg/ml respectively, p = 0.006). Following TLR-9 stimulation with CpG-B, monocyte cell cultures from ASD children produced significantly decreased levels of IL-1β (205.6 ± 45.7 vs. 584.8 ± 142.4 pg/ml, ASD vs TD, p = 0.01), IL-6 (9,601 ± 2,327 vs. 70,390 ± 29,050 pg/ml, p = 0.05), and TNFα (668.6 ± 211.0 vs. 2,184 ± 585.5 pg/ml, p = 0.02), GM-CSF (28.8 ± 7.3 vs. 71.4 ± 18.6 pg/ml p = 0.04) and MCP-1 (9,030 ± 1,811 vs. 23,010 ± 3,608 pg/ml p = 0.002) compared with TD controls (). CpG-B induced production of IL-8 (91,949 ± 16,653 vs. 101,946 ± 14,410 pg/ml, ASD vs TD) was not statistically different between monocyte cell cultures from ASD and TD control children. No differences were observed in cell surface expression of TLR 3, 4, 5 and 9 on CD14+ monocytes from children with ASD and TD controls (data not shown). In addition, induced cytokine levels following stimulation with TLR 2, TLR 3, TLR 4, TLR 5 and TLR 9 ligands were not associated with concentrations of TH1 (IL-12p70, IFNγ) or TH2 (IL-4, IL-5, IL-10) cytokines in plasma samples from the same children with ASD and TD controls as assessed by Luminex assays (data not shown).
Figure 4 Induced cytokine concentrations in monocyte cell culture supernatants following stimulation with the TLR 3 ligand, poly I:C (A), the TLR 5 ligand, flagellin (B) and the TLR 9 ligand, CpG (C). Responses are shown for supernatants from children with ASD (more ...)