Rheumatoid arthritis patients exhibit impaired Candida albicans-specific Th17 responses

Abstract

Introduction: Accumulating data implicate the CD4+ T cell subset (Th17 cells) in rheumatoid arthritis (RA). IL-17 is an inflammatory cytokine that induces tumor necrosis factor (TNF)α, IL-1β and IL-6, all of which are targets of biologic therapies used to treat RA. RA patients are well documented to experience more infections than age-matched controls, and biologic therapies further increase the risk of infection. The Th17/IL-17 axis is vital for immunity to fungi, especially the commensal fungus Candida albicans. Therefore, we were prompted to examine the relationship between RA and susceptibility to C. albicans because of the increasing interest in Th17 cells and IL-17 in driving autoimmunity, and the advent of new biologics that target this pathway.

Methods: We analyzed peripheral blood and saliva from 48 RA and 33 healthy control subjects. To assess C. albicans-specific Th17 responses, PBMCs were co-cultured with heat-killed C. albicans extract, and IL-17A levels in conditioned supernatants were measured by ELISA. The frequency of Th17 and Th1 cells was determined by flow cytometry. As a measure of IL-17A-mediated effector responses, we evaluated C. albicans colonization rates in the oral cavity, salivary fungicidal activity and levels of the antimicrobial peptide β-defensin 2 (BD2) in saliva.

Results: Compared to controls, PBMCs from RA subjects exhibited elevated baseline production of IL-17A (P = 0.004), although they had similar capacity to produce IL-17A in response to Th17 cell differentiating cytokines (P = 0.91). However RA PBMCs secreted less IL-17A in response to C. albicans antigens (P = 0.006). Significantly more RA patients were colonized with C. albicans in the oral cavity than healthy subjects (P = 0.02). Concomitantly, RA saliva had reduced concentrations of salivary BD2 (P = 0.02). Nonetheless, salivary fungicidal activity was preserved in RA subjects (P = 0.70).

Conclusions: RA subjects exhibit detectable impairments in oral immune responses to C. albicans, a strongly Th17-dependent opportunistic pathogen, despite an overall elevated baseline production of IL-17A.

Figure 1

Reduced Candida albicans-induced Th17/IL-17A responses in rheumatoid arthritis. Rheumatoid arthritis (RA) subjects exhibit impaired Candida albicans-specific T-cell responses compared with healthy controls. (A) Peripheral blood mononuclear cells (PBMCs) from RA subjects and controls were cultured for 5 days in media ± heat-killed (HK) C. albicans extract or T-helper type (Th)17 differentiating cytokines (interleukin (IL)-1β, IL-6, IL-23, anti-IL4 and anti-interferon gamma (anti-IFNγ)). IL-17A in cell free culture supernatants was measured by enzyme-linked immunosorbent assay (Student’s t test). (B) Tumor necrosis factor (TNF) alpha inhibitors do not exacerbate C. albicans-specific T-cell responses compared with disease-modifying anti-rheumatic drugs (DMARDs). PBMCs were cultured as in (A) with media alone, HK C. albicans extract or Th17 differentiating cytokines. IL-17A production was assessed in the indicated patient subgroups based on medication use (Mann–Whitney U test). (C) TNFα inhibitors do not alter the fraction of circulating Th17 or Th1 cells in RA subjects with controlled disease. PBMCs from RA subjects and healthy controls were gated on CD3⁺CD4⁺ lymphocytes and were analyzed for expression of IL-17A (Th17) and IFNγ⁺ (Th1) cells. Data were stratified by medication use (Mann–Whitney U test).

Figure 2

Rheumatoid arthritis subjects have a lower proportion of IL-17A⁺ but a higher proportion of IFNγ⁺ CD4⁺ T cells compared with controls. (A) Circulating T-helper type (Th)17 and Th1 cells. Peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients and healthy controls were gated on CD3⁺CD4⁺ lymphocytes and were analyzed for expression of interleukin (IL)-17A (Th17 cells) and interferon gamma (IFNγ; Th1 cells) (Mann–Whitney U test). (B) Circulating CD161⁺ and T_EM cells. PBMCs were gated on CD3⁺CD4⁺ lymphocytes and were analyzed for expression of CD161 and CD45RO (T_EM cells) (Mann–Whitney U test). (C) CD161⁺IL17A⁺ population. PBMCs were gated on CD3⁺CD4⁺CD161⁺ and analyzed for expression of IL-17A and IFNγ (Mann–Whitney U test). (D) T_EM IL-17A⁺ population. PBMCs were gated on CD3⁺CD4⁺CD45RO⁺ lymphocytes and were analyzed for expression of IL-17A and IFNγ (Mann–Whitney U test).

Figure 3

Rheumatoid arthritis subjects have impaired IL-17A-dependent anti-microbial peptide induction, but preserved oral immunity to Candida albicans. (A), (B)Candida albicans colonization in the oral cavity. Saliva (25 μl) from the indicated cohorts was analyzed for endogenous C. albicans colonization by plating on yeast peptone dextrose (YPD) agar in triplicate and performing colony enumeration. Data are presented as (A) the percent of each cohort that exhibited any oral colonization or (B) the number of colony-forming units (CFU) in each cohort (Fischer’s exact test and Mann–Whitney U test, respectively). (C) Rheumatoid arthritis (RA) subjects exhibit reduced salivary β-defensin 2 (BD2) levels. BD2 in saliva from the indicated cohorts was determined by enzyme-linked immunosorbent assay (Student’s t test). (D) RA subjects show intact salivary candidacidal activity. Salivary Candida killing was determined by centrifuging saliva to remove endogenous microbes and incubating the spun fraction with 10⁶C. albicans (reference strain CAF2-1) for 1 hour at 37°C. Triplicate samples were plated on YPD for colony enumeration and killing is indicated as the percentage of a PBS control (Student’s t test).