Cerebrospinal fluid cytokines in COVID-19: a review and meta-analysis

Abstract

Introduction

Neurological involvement can occur in patients with SARS-CoV-2 infections, resulting in coronavirus disease 2019 (COVID-19). Cytokine alterations are associated with neurological symptoms in COVID-19. We performed a review of cytokines in the cerebrospinal fluid (CSF) of patients with COVID-19.

Methods

Two reviewers independently searched PubMed for all relevant articles published prior to November 11, 2022. Active SARS-CoV-2 infection and CSF cytokine analyses were required for inclusion.

Results

Three-hundred forty-six patients with COVID-19 and 356 controls from 28 studies were included. SARS-CoV-2 PCR was positive in the CSF of 0.9% (3/337) of patients with COVID-19. Thirty-seven different cytokines were elevated in the CSF of patients with COVID-19 when compared to controls and the standards set forth by individual assays used in each study. Of the 37 cytokines, IL-6 and IL-8 were most commonly elevated. CSF IL-6 is elevated in 60%, and CSF IL-8 is elevated in 51% of patients with COVID-19.

Conclusion

Levels of several inflammatory cytokines are elevated in the CSF of patients with COVID-19, and SARS-CoV-2 PCR is often not isolated in the CSF of patients with COVID-19. Many patients with COVID-19 have neurological symptoms and given the cytokine elevations in the absence of detectable viral RNA in cerebrospinal fluid; further study of the CSF cytokine profiles and pathogenesis of neurological symptoms in COVID-19 is needed.

Introduction

Cytokines are signaling proteins and drivers of inflammation, including in the central nervous system (CNS) [1]. As an integral component of the immune response within the CNS, cytokines play a role in many different disease processes including infection, inflammation, and ischemic injury [1]. Hypercytokinemia, or cytokine storm, is implicated in encephalitis and encephalopathy in coronavirus disease 2019 (COVID-19) which is triggered by the virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [2]. Moreover, elevated cerebrospinal fluid (CSF) cytokines are also observed in patients with encephalitis and encephalopathy [3]. Release of large amounts of cytokines is a complication of infection that can be neurotoxic and, in some cases, fatal [4]. In COVID-19, infection from SARS-CoV-2 can cause systemic inflammatory response syndrome (SIRS), which can in turn cause elevated levels of cytokines that may be damaging to the nervous system [2].

Central nervous system involvement with a range of accompanying clinical and laboratory findings has been reported in cases of SARS-CoV-2 infection, and SARS-CoV-2 may use different mechanisms to infiltrate the CNS [5]. Encephalopathy and encephalitis are two manifestations of nervous system involvement commonly seen in COVID-19, and both can be driven by inflammation [5]. Many studies have analyzed CSF in COVID-19 patients with neurological symptoms, with varied levels of inflammatory cytokines found in CSF samples for actively infected patients. However, most of these studies have had very small patient populations. Few studies have investigated CSF cytokines in large populations of patients with COVID-19 [6]. This review aims to investigate the cytokine profile of CSF in COVID-19 in order to better characterize the CNS inflammation that results from SARS-CoV-2 infection as well as the utility of CSF cytokine analysis in patients with COVID-19.

Methods

IRB approval was not needed for this study. A review was conducted by two independent reviewers for manuscripts published on any date prior to November 11, 2022. The terms searched in PubMed included “CSF cytokines and COVID,” and “cerebrospinal fluid cytokines and COVID.” Publications reporting on levels of cytokines in the cerebrospinal fluid of patients with COVID-19 were included. Case reports and case series were included in the review. Whether cytokine levels were significantly elevated or not as compared to controls was defined by the assays used in each of the individual studies. Publications without full-text availability were excluded. Preprint publications and publications not in English were also excluded. Articles that were duplicated between the two search terms were only included once. Articles that were not primary sources were also excluded. Articles reporting on serum levels of cytokines in COVID-19 infection without analysis of cerebrospinal fluid were excluded, and articles reporting on results of CSF analysis in animal models or in patients without active SARS-CoV-2 infection were also excluded.

Data collected included the number of patients with COVID-19 who had CSF collected and analyzed, CSF cytokines investigated in each study, and number of controls for each study. The total number of patients with COVID-19 was calculated from all patients in the included studies who had CSF analyzed for cytokine levels. In addition, we recorded whether each cytokine was elevated or not in comparison with controls and in how many patients it was elevated. For each cytokine, we calculated the percentage of patients out of the total number of COVID-19 patients for which the specific cytokine was measured who had cytokine elevation. For each study, the number of patients for which CSF COVID-19 PCR was positive was also recorded.

Results

A total of 320 articles were identified, and 273 articles were screened after 47 duplicate articles were removed. Of the records screened, 187 were excluded based on examination of the title and abstract alone and determination that the articles were not relevant to this review. After screening, 86 full-text articles were reviewed, and a further 51 articles were excluded because they did not investigate the aims of this review. In addition, 7 publications that were not primary articles were removed. In total, 28 studies were included that were primary articles in keeping with the primary aims of this review (Fig. 1).

Fig. 1.

Flowchart of review of cerebrospinal fluid (CSF) cytokines in patients with COVID-19

Data from 346 patients with active COVID-19 infection and CSF analysis of cytokine levels were included. There were 356 total control patients in the 28 studies who underwent CSF analysis, though not every study included controls. Thirteen out of the 28 studies had no control patients, and cytokine elevation was determined by the limits set forth in the assay for each study. A positive COVID-19 PCR was detected in the CSF of 0.9% (3/339) of total patients with COVID-19 infection, although 3 of the 28 studies (7 total patients) did not report results of COVID-19 PCR in CSF. In the 346 patients included in this study, symptoms included: seizures [4, 6–14], encephalopathy [2, 4, 6, 7, 9–25], stroke [2, 6, 7, 12, 26], weakness [2, 4, 6, 14, 22, 27], paresthesias [2, 6, 14, 22], myelitis [2, 6, 25], Guillain Barre syndrome, [2, 6, 27] myoclonus [13, 19] headache [6, 25] and other (Psychiatric [8], vision changes with a new white matter lesion, [28] and neuromyelitis optica spectrum disorder [25]).

There were elevations in 37 different cytokines measured in the CSF of patients with COVID-19. IL-6 and IL-8 were elevated in the highest absolute number of patients with 60% (203/340) and 51% (138/270) of patients, respectively. Other cytokines with comparatively higher percentages of elevation included beta-2 microglobulin at 97% (56/58), IL-2 at 51% (82/160) and IL-18 at 53% (30/57). As for outcomes, certain cytokines were reported with poor outcomes, including β2-microglobulin [4], IL-1β [4], IL-6 [4, 7, 15], IL-8 [4, 21], and MMP-10 [12]. Moreover, neurofilament light chain (NfL) was also associated with poor outcomes [2, 4, 26, 29]. However, some of these elevated cytokines were reported in those with good outcomes, and in others, the outcomes were unknown (Table 1).

Table 1.

Grouped data of cerebrospinal fluid cytokines in 346 patients from all 28 publications. The outcomes that are reported (and not necessarily associations) with elevated cytokines are listed

Cytokine	Number with cytokine elevation/number tested	% Positive	Reported outcomes
			Poor	Good	Unknown
β2M	56/58	96.6	[4]	[4, 17, 29]
CXCL1	13/70	18.6			[12]
CXCL6 (GCP2)	13/48	27.1			[12]
CXCL11	13/48	27.1			[12]
CX3CL1	10/87	11.5		[22]
IFN-γ	14/238	5.9		[29]	[12, 24, 25]
IL-1	1/95	1.1		[18]
IL-1β	42/216	19.4	[4]	[4, 9, 22]	[16]
IL-2	82/160	51.3		[29]	[24, 25]
IL-4	15/160	9.4			[25, 28, 40]
IL-6	203/340	59.7	[4, 7, 15]	[2, 4, 10, 17–19, 22, 26, 29]	[6, 12–14, 20, 25, 27]
IL-8 (CXCL8)	138/270	51.1	[4, 21]	[2, 4, 10, 17, 23]	[6, 11–14, 16, 25, 27]
IL-10	75/179	41.9		[26, 29]	[13, 25]
IL-12	36/182	19.8		[9, 26]	[25]
IL-13	2/64	3.1			[24]
IL-15	23/85	27.1			[14]
IL-18	30/57	52.6		[2]	[2]
IL-23	1/27	3.7		[19]
IL-31	1/23	4.3		[19]
IP-10 (CXCL10)	30/156	19.2		[10]	[12, 13, 25]
MCP-1 (CCL2)	42/193	21.8		[2]	[2, 6]
MCP-2 (CCL8)	13/54	24.1			[12]
MCP-3 (CCL7)	10/85	11.8			[6]
MCP-5 (CCL12)	10/55	18.2			[20]
MDC (CCL22)	5/7	71.4		[8]
MIG (CXCL9)	18/111	16.2		[8]	[12]
MIP-1α (CCL3)	10/82	12.2			[13, 25]
MIP-1β (CCL4)	38/105	36.2		[8]	[14]
MIP-3α (CCL20)	13/48	27.1			[12]
MMP-10	13/48	27.1	[12]
NfL	42/158	26.6	[2, 4, 26, 29]	[8, 26, 29]	[16]
sgp130	1/1	100		[17]
TARC (CCL17)	5/5	100		[8]
TGL-b	1/86	1.2			[25]
TNL-α	100/258	38.8		[4, 17, 18, 29]	[6, 13, 16, 20, 25]
TNFRSF11b	14/35	40			[41]
EIF4EBP1/4E-BP1	13/48	27.1			[12]

β2M β2-microglobulin, IFN interferon, IL interleukin, IP interferon gamma-induced protein 10, MCP monocyte chemoattractant protein, CCL chemokine ligand, MIP macrophage inflammatory protein, MMP matrix metalloproteinase, NfL neurofilament light chain, TARC thymus and activation-regulated chemokine, TGF transforming growth factor, TNFRSF11B tumor necrosis factor receptor superfamily member 11B, EIF4EP1 eukaryotic translation initiation factor 4E-binding protein 1

Discussion

COVID-19, though primarily a respiratory illness, has a variety of neurological manifestations of infection including headache, stroke, encephalopathy, seizure, and olfactory disturbances [16, 30–34]. There is limited data concerning clinical and laboratory findings of neurological impairment in COVID-19, as well as the underlying pathophysiology [21]. This review found that several cytokines were consistently elevated in the CSF of patients with COVID-19, even in the absence of positive SARS-CoV-2 PCR in the CSF. Some of the most commonly elevated cytokines included IL-6 (59.7%), IL-8 (51.1%), and TNF-α (38.8%), all of which are proinflammatory cytokines.

Nervous system involvement in COVID-19

Neurological symptoms are present in as many as 35% of hospitalized patients with COVID-19. [30] Given the prevalence of neurological dysfunction in COVID-19, several mechanisms for entry of SARS-CoV-2 into the CNS have been proposed. These mechanisms include: viral passage by infecting of other cells that are able to cross the blood–brain barrier, transcellular migration facilitated by the ACE2 receptor, paracellular migration, via vascular endothelium, or by crossing the cribriform plate following entry through the olfactory system are several of the proposed mechanisms [5, 29, 31, 35]. Another theory is that pre-existing nervous system pathology could alter the blood–brain barrier and allow viral entry to the CNS. [26] Though there are several proposed theories for SARS-CoV-2 entry to the CNS, viral RNA is often not detected in the CSF of patients with active COVID-19. Out of the 339 patients with known CSF SARS-CoV-2 PCR results, only 3 patients mentioned here had a positive SARS-CoV-2 PCR from their CSF samples. While detection of viral RNA in the CSF of patients with COVID-19 is very infrequent and with alterations in cytokine profiles, the frequent neurological symptoms associated with COVID-19 suggest that there is an immune response to the virus occurring in the CNS [12].

Systemic versus intrathecal cytokines

In COVID-19 patients with neurological complications, elevated CSF cytokines could either be due to systemic versus intrathecal specific cytokines. Consistent with the theory that blood–brain barrier dysfunction could allow entry of the virus into the CNS, blood–brain barrier breakdown would allow for systemic cytokines to increase CSF cytokine levels [16]. Systemic cytokine profiles are complex and differ among patients with COVID-19. Cytokines can be divided into six families: interleukins (ILs), tumor necrosis factors (TNFs), interferons (IFNs), hematopoietic growth factors, chemokines, and transforming growth factor-β (TGF-β). However, the proinflammatory cytokines TNF-α, IL-1, and IL-6 are generally elevated in COVID-19 infection, including higher levels in those with severe as compared to mild COVID-19 infection [36]. The interferon classes include types I, II, and III. Type I interferons, such as IFN-α and IFN-β, have anti-viral properties, although type I IFN therapy has shown no efficacy. Hematopoietic growth factors, including granulocyte colony-stimulating factors (G-CSFs) and GM-CSFs, are implicated in cytokine storms in COVID-19. TNFs are generally high in COVID-19 and affect IL-1 and IL-6 levels. Conversely, TGF-β is a multifunctional immunoregulatory cytokine that can inhibit the immune response (e.g., induces regulatory T cells and inhibit B cell proliferation) but also generate proinflammatory T cells (e.g., T helper 17 cells). For additional details on systemic cytokines in COVID-19, see this review [36].

Many studies in this review reported serum cytokine elevations along with the elevations seen in the CSF, though the cytokine profiles often varied between serum and CSF. This review did not take into account measurements of blood–brain barrier breakdown which would help assess whether cytokine elevations were simply a result of systemic cytokines or intrathecal production. One study of six patients with COVID-19 found evidence of CNS-specific inflammation as opposed to systemic inflammation by comparing the CSF to serum cytokines [9]. Another study compared the CSF cytokines in COVID-19 patients to healthy controls and controls with non-COVID-19 neurological disorders and found similar cytokine profiles (in particular IL-6, TNFα, IL-12p70, and IL-10) in COVID-19 versus other non-COVID-19 neurological controls [26].

Cytokines detected post-SARS-CoV-2 vaccination have been reported in a few cases (see review [37]). One patient with encephalopathy post-SARS-CoV-2 vaccination had elevated IL-6 in both serum and CSF, with elevated IL-8 only in the CSF [38]. Another patient who presented with rhomboencephalitis had elevated IL-8 in both serum and CSF, with elevated IL-10, IFN-g, and IL-6 in the CSF [39].

Cytokine profile of neurosymptomatic patients and outcomes

The inflammatory cytokine IL-6 is considered an integral part of the cytokine storm that may damage the blood–brain barrier and allow CNS penetration by viral infections, which could then lead to the neurological symptoms seen in COVID-19 [3]. CSF IL-6 [20, 29], IL-8 (CXCL8) [2, 6], TNF-α [20, 29], and IFN-γ [29] are commonly elevated in those with COVID-19 and neurological symptoms as compared to those without neurological symptom or healthy controls.

CSF IL-6, TNF-α, IL-10, and IL-12p70 were elevated in COVID-19 related stroke compared to healthy controls. However, when the COVID-19 stroke group was compared to stroke patients without COVID-19, no differences in cytokines were observed, suggesting that the elevation in cytokines may have been driven by stroke instead of by COVID-19 infection [26]. Another study categorized CSF profiles based on the neurological symptoms of patients with COVID-19 and the CSF profiles could differentiate between inflammatory syndromes, encephalopathy, and headache. For example, increased IL-6, CXCL8, and CXCL10 levels were observed in inflammatory syndromes and elevated CSF IL-2 in encephalopathy [25]. Moreover, a separate study compared patients with COVID-19 and encephalopathy to healthy controls and encephalopathic patients without COVID-19. The pattern of cytokine elevation with early increases in IL-8 for COVID-19 with encephalitis was unique [4]. Identifying these unique patterns may be useful in order to support clinical diagnoses of neurological syndromes with laboratory findings [25].

Limitations

Many of the studies included in the review were case reports or case series with a limited number of patients and often those studies did not include healthy controls for comparison. In addition, given the invasive nature of CSF collection, many patients were often only included in the studies because they had neurological symptoms warranting a lumbar puncture. Moreover, not all patients were tested with the same cytokine panels or the same assays, which may introduce bias. Many of the patients with COVID-19 who have neurological symptoms are more severely ill as well, which could have an effect on the laboratory values [29]. Stratification by age was also challenging. While the lack of comparison to controls as well as small population size for many of the studies is a limitation of the review, we have complied a comprehensive list of cytokines reportedly elevated in CSF of those with COVID-19 and neurological symptoms.

Conclusion

Here, we provide a list of cytokines elevated in the CSF of patients with neurological symptoms in the setting of COVID-19. Inflammatory cytokines including IL-6, IL-8, and TNF-α were consistently elevated in the CSF of patients with COVID-19 across the studies examined in this review. IL-2, beta-2 microglobulin, and IL-18 were also consistently elevated. At the same time, viral RNA was consistently undetectable in the cerebrospinal fluid of patients with confirmed cases of COVID-19 across studies. Neurological involvement is common in COVID-19, and the presence of inflammatory cytokines in the CNS with concurrent absence of COVID-19 viral RNA in the CNS points to a need for further to better understand how the parainfectious inflammatory response to SARS-CoV-2 infection causes neurological impairment. Additionally, limited data are available in children, with only one study on children available currently [8]. Moreover, the role of cytokines and persistent inflammation in post-acute sequelae SARS-CoV-2 infection (PASC), also known as long-COVID, should also be explored. Further investigation of CSF cytokine profiles in COVID-19 for larger patient populations including healthy controls and patients without neurological symptoms would be warranted.