NSAIDs affect dendritic cell cytokine production

Abstract

Background: Immunotherapy is now considered as the new pillar in treatment of cancer patients. Dendritic cells (DCs) play an essential role in stimulating anti-tumor immune responses, as they are capable of cross-presenting exogenous tumor antigens in MHCI complexes to activate naïve CD8+ T cells. Analgesics, like non-steroid anti-inflammatory drugs (NSAIDs), are frequently given to cancer patients to help relieve pain, however little is known about their impact on DC function.

Methods: Here, we investigated the effect of the NSAIDs diclofenac, ibuprofen and celecoxib on the three key processes of DCs required for proper CD8+ cytotoxic T cell induction: antigen cross-presentation, co-stimulatory marker expression, and cytokine production.

Results: Our results show that TLR-induced pro- and anti-inflammatory cytokine excretion by human monocyte derived and murine bone-marrow derived DCs is diminished after NSAID exposure.

Conclusions: These results indicate that various NSAIDs can affect DC function and warrant further investigation into the impact of NSAIDs on DC priming of T cells and cancer immunotherapy efficacy.

Fig 1. COX expression and PGE2 production upon TLR exposure.

mBMDCs were treated for 6 hr (a) and 18 hr (a, b) with the TLR adjuvants LPS, CpG, and R848. (a) RT-QPCR was performed for mRNA expression of COX1 and COX2 (mBMDCs, n = 6). (b) PGE2 was measured using a competitive enzyme immunoassay in PCA-treated supernatant (n = 3). Results are shown as means with SEM. Statistical significance was calculated using a one-way ANOVA with Dunnett’s multiple comparison test, medium versus rest.

Fig 2. NSAIDs do not affect ISCOM induced cross-presentation.

(a) Timeline shows the experimental setup for assessing cross-presentation, maturation markers and cytokine production by mBMDCs. (b-d) mBMDCs were first pretreated with NSAIDs for 6 hr, washed and subsequently treated with OVA protein and ISCOMs for 2.5 hr, and then co-cultured with B3Z T cells for 18 hr. As a positive control for viability and MHC-I levels, mBMDCs were pulsed with OVA peptide (SIINFEKL) 0.5 hr before coculture with B3Z T cells (n = 4). ISCOM induced cross-presentation of OVA protein and stable loading of exogenous SIINFEKL by celecoxib (b), diclofenac (c), and ibuprofen (d) pretreated DCs. Results are shown as means with SEM. Statistical significance was calculated using a one-way ANOVA with Dunnett’s multiple comparison test, medium versus rest.

Fig 3. NSAIDs do not alter TLR-induced maturation marker expression.

(a-d) mBMDCs were first pretreated with NSAIDs for 6 hr, followed by a 2.5 hr TLR stimulation with LPS, CpG, or R848. After overnight rest in fresh medium, composition of the mBMDC culture (a, b) and maturation marker expression (c, d) were analyzed using flow cytometry (n = 4). Results are shown as means with SEM. (a) Percentage CD11c+ cells in BMDC culture. (b) GM-DC/GM-MAC ratio within CD11c+ population. (c) Mean Fluorescent Intensity (MFI) ± SEM of CD80 and (d) CD86 in CD11c+ population. Statistical significance was calculated using a one-way ANOVA with Dunnett’s multiple comparison test, medium versus rest.

Fig 4. NSAID preincubation reduces TLR induced cytokine production.

(a-c) mBMDCs were first pretreated with NSAIDs for 6 hr, followed by a 2.5 hr TLR stimulation with LPS, CpG, or R848. After overnight rest in fresh medium, cytokines were measured using ELISA (n = 4). IL-10, IL-12, TNF-α, and IL-6 production after TLR stimulation by (a) celecoxib, (b) diclofenac, and (c) ibuprofen preincubated DCs. Results are shown as means with SEM. Statistical significance was calculated using a one-way ANOVA with Dunnett’s multiple comparison test, medium versus rest.

Fig 5. Decreased cytokine production by moDCs upon TLR stimulation when preincubated with NSAIDs.

(a-c) moDCs were first pretreated with NSAIDs for 6 hr, followed by a 2.5 hr TLR stimulation with LPS or R848. After overnight rest in fresh medium, cytokines were measured using ELISA (n = 5–6). IL-10, TNF-α, and IL-6 production after TLR stimulation by (a) celecoxib, (b) diclofenac, and (c) ibuprofen preincubated moDCs. Results are shown as means with SEM. Statistical significance was calculated using Mixed-effects analysis with Dunnett’s multiple comparisons test.