SARS-CoV-2 primarily causes a respiratory tract infection and is associated with increased circulating proinflammatory cytokines and inflammatory mediators, with a potential progression to cytokine storm (1, 16). Though assumed by Bolay et al (2, 9), it had not been shown so far whether inflammatory molecules were associated with COVID-19 headache. We studied peripheral circulating inflammatory molecules in COVID-19 patients with moderate severity hospitalized in the routine ward. Presence of severe headache in COVID-19 was associated with elevated inflammatory and/or nociceptive molecules like HMGB1, NLRP3, IL-6, and ACE2 compared to the non-headache COVID-19 sufferers, while anti-inflammatory cytokine levels of IL-10 did not show any difference at hospital admission between the two groups. Moreover, NLRP3 and HMGB1 were correlated with headache duration and paracetamol unresponsiveness respectively. Robust inflammatory response was detected particularly in COVID-19 patients with severe headache. In supporting, these patients were also associated with increased frequency of pulmonary involvement but not needing any ICU management.
Angiotensin-converting enzyme 2 (ACE2) has gained importance in COVID-19 pathogenesis because membrane-bound ACE2 is the receptor for SARS-CoV-2 cellular entry (2, 9, 17). ACE2 is widely expressed in the human body, including epithelial cells in nasal and oral mucosa, pneumocytes in the respiratory system, besides the vascular endothelial cells and smooth muscle cells (18). Soluble ACE2 on the other side could reflect the cleavage of membrane-bound ACE2 by the SARS-CoV-2 entry and lysis of ACE2-expressing cells. High levels of plasma ACE2 was proposed to indicate poor outcome in critically ill patients (19, 20). The moderately affected disease process was different in our patients, in whom elevated ACE2 levels were seen in association with good outcomes without any ARDS, serious complication or mortality. Increased circulating ACE2 level was even suggested to denote endogenous protection against SARS-CoV-2 (21, 22).
Angiotensin II levels detected in our study merit particular emphasis. Unless cleaved by ACE2 enzyme, angiotensin II peptide mediates various functions including regulation of blood pressure, vascular tonus, electrolyte balance, and induce inflammation, produce reactive oxygen species and activate NLRP3 inflammasome (2, 9, 17, 23, 24). The internalization of membrane bound ACE2 by the virus binding would result in the unbalanced activity of angiotensin II, yielding NLRP3 inflammasome activation and release of pro-inflammatory cytokines subsequently recruiting inflammatory cells (2, 9, 18, 23). Angiotensin II is also involved in pain signaling in the trigeminal ganglia (25) and in inducing nociceptive behavior (26). Besides, angiotensin II receptor inhibitors are effective prophylaxis drugs for migraine headache (27) and implicated in neuropathic pain treatment (28). Consequently, elevated circulating angiotensin II levels in COVID-19 patients with severe headache were assumed to contribute to trigeminal nociception and indicate increased activity of inflammatory cascade via its receptors (2, 9). However, our study failed to show elevated serum levels of angiotensin II in COVID − 19 patients with headache. The circulating angiotensin II levels were significantly decreased in patients with COVID-19 headache on the contrary to elevated ACE2 levels. Increased circulating ACE2 may cleave angiotensin II and be responsible for the reduced angiotensin II at the point of our measurements in COVID-19 patients. Decreased serum angiotensin II peptide levels were already shown in COVID-19 patients (29). Taken all together, our results may suggest that decreased circulating angiotensin II is implausible to be responsible for the headache in COVID-19 patients. Likewise, comparable systemic circulating CGRP levels in COVID-19 patients with or without headache groups in our study lessens the role systemic CGRP as a potential trigger for COVID-19 related headache. That was in line with the recent study that reported lower serum CGRP levels in COVID-19 patients (30).
Elevated HMGB1 level is significantly associated with the emergence of severe headache and correlated with paracetamol unresponsiveness in our patients with COVID-19 headache. HMGB1 is a prototypical damage-associated molecular pattern (DAMP) representing a critical marker of intense inflammation and studies have shown that serum HMGB1 is increased in COVID-19 cases and positively correlated with disease severity. Moreover, serum HMGB1 also decreases when patients improved (31, 32). HMGB1 may be involved in the cytokine storm or as a second way also by ACE2 expression in alveolar epithelial cells, in the COVID-19 pathogenesis. Increased levels of serum HMGB1 are considered a reliable marker for systemic inflammation but do not indicate the specific tool initially involved in the inflammatory disease. Additionally, HMGB1 regulates autophagy, which is related to SARS-CoV-2 entry. The complex role of HMGB1 in migraine headache was reviewed in detail (33). HMGB1 can cause nociception via direct activation of receptor for advanced glycation end-products (RAGE) in sensory neurons (34). HMGB1 is also implicated in maintaining the neuropathic pain state and trigeminal neuropathic pain. Treatment of the trigeminal nerve with anti-HMGB1 neutralizing antibody prevented pain behavior and blocked macrophage and microglia activation (35). Therefore, HMGB1 could be a key player in the development of severe and long-lasting COVID-19 headache. Our results indicated that it will be important to study the potential role of HMGB1 in COVID-19-related headache in related models to gain more insight into the headache mechanisms triggered by external sources, specifically COVID-19.
In COVID-19, SARS-CoV activates NLRP3 inflammasome, which is critical in the innate immune defense system, yielding proinflammatory cytokines including IL- 6, prostaglandins, and leukotrienes to amplify tissue inflammation (9, 23, 24, 36). Recently, the possible role of NLRP3 inflammasome were implicated in the development of headache in COVID-19 (9). NLRP3 was significantly associated with headache in COVID-19 and correlated with duration of headache and hospital stay, in our study. In glyceryl trinitrate-induced experimental headache studies, activation of NFkb, IL1β, IL-6 and NLRP3 inflammasome were shown in dural macrophages and in the brainstem microglia (37–39). However, the exact role of circulating NLRP3 levels in inducing trigeminal nociception needs to be determined by further studies.
Development of headache represents a nociceptive activation of the trigeminal nerve through the neuro-inflammatory-vascular processes, where release of CGRP from perivascular trigeminal nerve endings, vascular reactivity and inflammation take place (2, 9, 40). Pro-inflammatory cytokine IL-6 was demonstrated to activate perivascular trigeminal nociceptors in the dura mater and induce headache in experimental studies (41, 42). Additionally, IL-6 was reported to provoke CGRP release particularly under heat conditions (43). Therefore, higher circulating levels of IL-6 in patients with more intense headache could point out a potential trigger role of IL-6 in COVID-19 headache.
Pulmonary involvement was detected by thorax CT in half of all patients and 65% of the COVID-19 headache sufferers. Intriguingly, D-dimer levels were significantly higher in patients with headache compared to non-headache sufferers. Increased serum D-dimer has been considered to reflect coagulopathy and poor prognosis in severe COVID-19 patients (44), but we observed no thrombotic event or mortality in our study group. In COVID-19 patients with moderate disease severity, higher levels of D-dimer and IL-6 with increased frequency of pulmonary infiltration were associated with severe headache (5). As proposed by Hunt & Levi, D-dimer levels could represent the degree of lung inflammation, like other acute-phase proteins in COVID-19 (45). The latter would provide a rational explanation for the correlation of D-dimer elevation and pulmonary infiltration in our COVID-19 patients with headache. Moreover, the presence of headache in hospitalized COVID-19 patients was stated as an independent predictor of lower risk of mortality (46). Our results are different, as COVID-19 patients with hypoxia and who required ventilation in the ICU were not included in our study group. Yet, 35% of the patients without any pulmonary infiltration (Table-3) manifested severe headache intensity and elevation of IL-6, HMGB1. It seems that circulating inflammatory cytokines could also be associated with headache without pulmonary inflammation detected by CT.
We think that the headache correlated with the presence of pulmonary infiltration cannot merely be attributed to hypoxia as severe hypoxemia and/or hypercapnia were not identified in any of our patients. Rather, we propose pro-inflammatory mediators and cytokines released extremely during pulmonary infiltration could play a key role in the development of headache in the COVID-19 course. A recent study revealed that IL-6 levels were positively correlated with VAS scores in COVID-19 patients with headache (5). Also, ROC curve cut-off values were reported to indicate a moderate increase of IL-6 for defining COVID-19 headache (5). Both IL-6 and IL-10 are reported to be predictors of COVID-19 severity (47). The discordance of these cytokines, we found in COVID-19 patients with versus without headache, deserves special attention. IL-10, a typical anti-inflammatory cytokine did not show any difference in relation to the presence of headache, whereas IL-6, a major pro-inflammatory cytokine showed significantly higher levels in COVID-19 patients with headache, intriguingly. Even though IL-6 and IL-10 were noted as predictors for COVID-19 deterioration, all of our patients were followed up in the hospital but not in ICU. The similarity of the IL-10 levels could be due to the early sampling at the hospital admission, its course would be different during the COVID-19 process.
Our clinical study has some constraints, as the study was conducted in hospitalized patients in the general ward, without including patients in the ICU and non-hospitalized mild symptomatic patients. In future studies, other groups with COVID-19 headaches like those with significant comorbidities, those transferred to the ICU, and subjects with mild/moderate (VAS ≤ 7) headaches should be investigated prospectively to see the clinical relations and changes of the cytokines over time. Another potential limitation was that our results may indicate more severe involvement in cases with headache in comparison to those without and may reflect recruitment bias. A limited number of inflammatory molecules were studied at hospital admission and follow-up data providing their temporal progression during COVID-19 headache is lacking.
This pioneering study on headache in COVID-19 has several implications. So far, it is the first clinical study investigating the role of circulating inflammatory and nociceptive molecules such as HMGB1, NLRP3 inflammasome, IL-6, IL-10, angiotension II, and ACE2 in COVID-19 headache. Elevated HMGB1, NLRP3 and IL-6 levels could be discriminative for COVID-19 headache and may have implications for secondary headaches related to other systemic viral infections. No significant association of serum CGRP level with COVID-19 related headache is notable and rise the importance of HMGB1, NLRP3, IL-6 in COVID-19 headache pathogenesis. NLRP3 was correlated with headache duration and hospital stay, while paracetamol response was negatively associated with HMGB1 levels and positively correlated with IL-10 levels. Moreover, the current study conducted during a different wave of the pandemic confirmed the previous report that higher IL-6 levels and more frequent pulmonary infiltration were detected in COVID-19 patients with headache.