Cytokine Autoantibodies Are Associated with Infection Risk and Self-Perceived Health: Results from the Danish Blood Donor Study

Cytokines are pivotal signaling molecules in the immune system balancing pro- and anti-inflammatory immune responses [1]. There is emerging evidence that naturally occurring cytokine-specific autoantibodies (c-aAb) may critically influence immune competence [2‐5].

The influence of c-aAb on the systemic cytokine networks remains mostly unknown, though multiple studies have linked specific c-aAb to various pathologies and opportunistic infections [6‐8]. Most notably, granulocyte-macrophage colony-stimulating factor (GM-CSF) c-aAb have been identified as a causal factor for pulmonary alveolar proteinosis (PAP), a rare and debilitating respiratory disease [9, 10]. A range of c-aAb have also been labeled as phenotypic classifiers for various primary immunodeficiencies (including GM-CSF c-aAb for PAP) by the international union of immunological societies [11]. Conversely, c-aAb against pro-inflammatory interleukins (IL) have been associated with improved prognosis or reduced symptoms in some autoimmune diseases, such as rheumatoid arthritis and Sjögrens syndrome indicating that ultimately the biologic effect of c-aAb depends on the specific pathological context combined with the c-aAb specificity [12, 13].

The trigger for c-aAb production is debated. Some studies indicate that the genetic framework may promote c-aAb (e.g., specific variants of the antigen-presenting human leukocyte antigen or mutations of the autoimmune regulator gene, AIRE), in addition to microbe-induced loss of tolerance, through mechanisms such as molecular mimicry [14, 15].

We have previously reported that detectable levels of c-aAb are common among healthy individuals, with high levels being associated with older age and male sex. Furthermore, in healthy individuals, high levels of IL-6 c-aAb are associated with low C-reactive protein (CRP) levels, indicating a possible functional effect of naturally occurring, high-titer c-aAb [16].

The aim of the present study was to investigate the association between available indicators of c-aAb-mediated immune modulation, evidenced by infectious risk and self-perceived health. We hypothesized that high-titer levels of c-aAb may inflict immunomodulation to a degree that would either increase or reduce infectious risk and self-perceived health. Thus, we linked our previously c-aAb screened cohort of healthy blood donors to health register and questionnaire data to assess possible outcomes of c-aAb-mediated immunomodulation, with antimicrobial prescription filings and self-perceived health as endpoints. To our knowledge, this is the largest study of the biologic and potentially immunomodulatory effects of c-aAb in a healthy population to date.

Cytokine autoantibodies are critical players in both immunodeficiencies and autoimmune disease, inflicting a variety of pro- or anti-inflammatory effects, depending on the context and their specificity. The present study investigated the association between different c-aAb directed against pro- and anti-inflammatory cytokines in healthy individuals and infectious risk, assessed as time until the next prescription of antimicrobials. We found that high-titer levels of c-aAb against pro- and anti-inflammatory cytokines predicted increased and reduced risk, respectively, of prescriptions of various antimicrobials as a proxy for infectious risk. As we observed positive associations between the c-aAb MFI signals, we also studied the pairwise association of c-aAb with prescriptions, which suggested a pattern of cumulative c-aAb effects. These findings suggest that c-aAb in healthy individuals are present in levels that may have immunomodulatory effects, and in the following, we speculate on the possible mechanisms of these.

In line with prior studies [16], we found c-aAb levels to be associated with sex and age, as well as a novel association of IL-6 c-aAb and oral contraceptives. As such, we observed a strong sex interaction for c-aAb and our investigated outcomes. In women, high levels of IFNα c-aAb were associated with increased risk of macrolide, sulfonamide, and antibacterial prescriptions in general. Previous studies have associated IFNα c-aAb levels with the occurrence of viral infections and have established a strong genetic component for the occurrence of this c-aAb [26‐29]. This makes it a prime candidate for further studies as a risk factor for infection. The finding in the present study that INFα c-aAb levels associate with various antimicrobial prescriptions (indicative of different infections, i.e., respiratory infections and urinary tract infections) indicates a broader biologic effect of IFNα c-aAb though causality remains to be established. GM-CSF c-aAb have also been associated with multiple variants of opportunistic infections [30], and here we observed high levels of c-aAb to be associated with an increased risk of sulfonamide prescriptions in women. Interestingly, despite the previously observed association between IL-6 c-aAb and low levels of CRP [16], high levels of IL-6 c-aAb did not predict higher or lower risk of filing prescriptions in this study, which may reflect the pleiotropic roles of IL-6 as both a pro- and anti-inflammatory cytokine [31]. Furthermore, c-aAb have in some reports been observed to act as agonists of their target cytokine function [32], adding further complexity to our findings.

Notably, high levels of IL-10 c-aAb were associated with lower risk of penicillin and general antibacterial prescriptions in women, also when combined with other c-aAb. It may be speculated that the supposedly protective effects of IL-10 c-aAb may be due to suppression of the anti-inflammatory regulator IL-10, hereby creating a more pro-inflammatory and more potent antimicrobial environment, as previously reported [3].

As an alternative outcome to approximate c-aAb influence on participant health, we investigated the association to self-reported PCS and MCS health scores, hypothesizing that participants with c-aAb-mediated immunomodulation may present with lower or higher scores. Notably, we observed the same pattern for self-reported health scores as we did for antibacterial prescriptions, so women with a high level of IL-10 c-aAb had increased odds of high (good) PCS scores, again suggesting a potentially protective and positive effect of IL-10 c-aAb. It should be emphasized that the effects of IL-10 c-aAb are likely highly context-specific, as another previous study from our research group reported that IL-10 c-aAb were associated with increased risk of major adverse cardiovascular events (MACE) in kidney-transplanted patients, an outcome that is intrinsically linked to a pro-inflammatory environment [33]. Thus, though protective in regard to infections in healthy individuals, IL-10 c-aAb may constitute a risk factor for patients in whom excess inflammation may tip a balance towards adverse events such as organ transplant patients and autoimmune disease in general, again due to the cytokines’ many immunoregulatory functions [34, 35]. Conversely, c-aAb against pro-inflammatory cytokines IFNα and IL-1α have been linked to improved prognoses in autoimmune disease [6].

The observed associations between IL-10 c-aAb, prescriptions, and self-reported health being specific to women are notable, considering that women in our study had lower levels of detectable IL-10 c-aAb compared to men [16]. This finding may suggest a higher tolerance for IL-10 c-aAb in men. Conversely, we observed an association between high levels of IL-1α c-aAb and a low (poor) MCS in men, indicating increased sensitivity for this c-aAb in men. It should be noted that the significant effects were only observed for the highest levels of c-aAb, suggesting that these titers are a requirement for possible functional cytokine suppression and ensuing biologic effects.

The limited association between several high-titer c-aAb and infection or health-related outcomes may emphasize the redundancy of the human cytokine network, as c-aAb-mediated functional inhibition of just one cytokine may not be enough to challenge immune competence. Though there was a positive association between c-aAb, this seemed limited to their intermediary levels at most, with a near-total lack of association between high-titer c-aAb in individuals. This combined with their rarity means that we were unable to investigate cumulative incidences of high c-aAb levels as predictors of infection risk in this study. However, we assessed c-aAb overlap between the low/intermediary/high levels, and in women, we observed distinct patterns of higher or lower predicted risk of antibacterial prescriptions. The presence of intermediary or high levels of c-aAb against anti-inflammatory cytokine IL-10 and pleiotropic IL-6 coincided with reduced risk of antibacterial prescriptions, which may again be due to promoting an increasingly pro-inflammatory environment. Conversely, participants with high/intermediary levels of pro-inflammatory specific c-aAb had increased risk of antimicrobial prescriptions, suggesting additive immunosuppression by presence of several c-aAb against pro-inflammatory cytokines. Together, these data suggest that there may be cumulative immunomodulatory effects of c-aAb, depending on the specificity of the high-titer c-aAb present.

Overall, the data from the present study suggest that high levels of c-aAb mediate target-specific immunomodulation in healthy individuals. This, along with the many studies demonstrating associations between c-aAb and opportunistic infections [6, 8, 28, 30], raises further concern for the influence of high levels of c-aAb in immunocompromised individuals. Future studies in such patients are warranted to determine the impact of high levels of c-aAb on the immune system and overall patient health, as the influence of c-aAb on patient outcome which may be very different in this context. Given that immunological profiling, including cytokine assessment in plasma and stimulated cell cultures, is increasingly used as a precision medicine approach to improve care of immunocompromised patients, the present study supports the notion that monitoring c-aAb may be a critical component of such a profile.

In terms of strengths and limitations, this is to our knowledge the first study that investigates the association between cytokine autoantibody levels and infection risk and general health in healthy individuals. Because the study is cross-sectional and population-based and because outcomes are collected systematically and through public registries, we believe there are no systematic biases that could have interfered with the internal validity of the study. Though the use of a healthy and highly characterized blood donor cohort minimizes potential confounding effects of chronic illness or comorbidity, the generalizability of the results might be reduced because blood donors are generally healthier and have a healthier lifestyle as compared with the general population [36], which may result in higher than average self-perceived health scores compared to the general population [37]. Furthermore, it is critical to emphasize that the results of the study are purely based on associations and not causal relationships, which should be taken into account when interpreting the results. The number of analyses performed may also create a risk for Type I errors through multiple testing. Finally, there is also the possibility of systemic biases in the case of a self-reported outcome such as BMI, where weight may be underreported.

In conclusion, we demonstrated that high levels of c-aAb specific for several pro-inflammatory cytokines predicted increased risk of infection in healthy individuals, whereas high levels of c-aAb against anti-inflammatory IL-10 predicted lower infection risk, as well as increased self-reported physical health.

These findings indicate that c-aAb in healthy individuals are present in levels that may have immunomodulatory effects, depending on the specific c-aAb-targeted cytokine. Overall, the findings emphasize that high-titer cytokine autoantibodies are biologically active in healthy individuals.