Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are autoimmune disorders of unknown etiology. The prevalence of SLE and RA is 5.14 and 0.75 per 100,000 person per year respectively.1 Systemic conditions in SLE can exhibit arthritis as comorbidity whereas RA is limited to specific regions of the body. Common susceptible factors are known to escalate both the pathologies, but may result in the development of different disease conditions. Immunobiology of both disorders interrelate which reveals the interplay of different immune cells and cytokines. Since the level of multiple cytokines increased among both the pathologies, therefore it is very difficult to differentiate the patients of both disorders on basis of their cytokine profiles. Thus, understanding the complex play of cytokine levels among these patients with different disease severity states can be of importance in classifying as well as in designing their therapeutic regimes.

SLE is a prototype of systemic autoimmune condition where the increased level of various cytokines can be observed in saliva, urine, and in various organs. The level of IL-2 was diminished in the lupus patients and anti-IL-2 antibodies were also observed to be associated with disease activity.2 IL-38 level was positively correlated with diseases activity and negatively correlated with C3 and C4 protein level.3 The level of IL-12 correlated positively with SLEDAI and could also divert the T cell development towards the Th1 lineage. IL-23 diverts the T cell development towards Th17 cells which leads to the secretion of IL-17, found to be coupled with lupus nephritis. A number of chemokines were also observed to have potent role in modulating the severity in SLE. CCL2/MCP-1, CCL11/eotaxin, CXCL1, CXCL8/IL-8, CXCL10/IP-10, CXCL12/SDF-1 levels in lupus patients were found to be coupled with kidney manifestation and also correlated with CNS manifestations.4 Heterogeneity among cytokine and chemokine levels in lupus patients can make it difficult to predict them as biomarker(s) and hence the need for multiplexing and further clustering of cytokines based on different disease stages.

Rheumatoid arthritis (RA) is a systemic disease, causing autoimmune response leading to joint damage and lifelong disability in severe conditions. RA can also exhibit in two states: active or inactive which can be importantly modulated by the interaction of cytokines. The two arms of immune system are facilitated by IFNs that modulate severity of RA. TNFs shows the dual role in RA pathology as it can protect against arthritic condition when secreted by the T cell or it can flare the conditions when secreted by the B cells.5 TNFs and IL-17 up regulations can further amplify the expression of various effector molecules such as CXCL1, CXCL2, CXCL3, IL-6, IL-8, and MMP-3.6 IL-6 also triggers the secretion of VEGF which is an important cytokine in RA inducing angiogenesis and immune cell migration into joints triggering inflammatory cascade. IL-17 can also trigger the secretion of RANKL, which is crucial for osteoclast differentiation. Inflamed joints have elevated level of RANKL which in turn increases the osteoclast differentiation and development of bone erosion in RA.7 Understanding of the complex cytokine network in RA will provide the insight into pathological mechanisms as well as highlight novel biomarkers of diagnostic importance.

Cytokine inhibitors such as those against TNFs and IL-6 have already proved to be of clinical importance for both the pathologies. GM-CSF inhibitors were also employed for better outcomes among RA patients. Recently, the treatment strategies are modifying by shifting towards the disease-modifying anti-rheumatic drugs (DMARDS) which have potential to inhibit the intracellular signalling induced by the extracellular cytokines. However, studies showing the complex interplay of the various cytokines creates the urgent need of a therapy with multiple targets for the cytokines. Advancement in molecular biology techniques such as multiplexing enables us in understanding of functional interaction among cytokines at different disease states. Several recent studies have also employed such strategies to understand the functional and correlational interaction among cytokines from autoimmune patients. In this study, we analysed 40 analytes including cytokines and chemokines among healthy control (HC), lupus patients (SLE) and arthritis patients (RA). Alterations in the cytokine profiles were compared between HC group vs SLE or RA. Arthritis comorbidities among SLE patients were also compared with RA patients. The functional and correlation interactions among cytokines in SLE and RA patients might provide the important diagnostic evidence as well as abnormal interactions within cytokine profiles.

The outcomes obtained pointed towards the importance of cytokines profiling in differentiating the SLE and RA patients from healthy controls. Many cytokines emerge out as potential biomarkers which can differentiate the disorder on the basis of severity and comorbidities. It is crucial to note that the all patients involved in this study were on the therapies which can affect the immune response including cytokines levels. Beside this, we have observed the significantly altered level of cytokines among SLE patients including the increased level of SDF-1a/CXCL12, IL-18, IP-10/CXCL10, MIF, M-CSF, SCF which was also in accordance with the literature.8 The decreased Gro-a/CXCL1 and IL-9 levels in SLE was in oppose to literature where the Gro-a and IL-9 were considered as potential marker for diseases activity. The decreased RANTES and MIP-1b level was also in contrast to literature where the levels of both was positively correlated with diseases severity in SLE.9 This decrease among pro-inflammatory cytokines in lupus patients might be due to their daily medication. Elevated IL-18 level among SLE patients known to activates the Th1 and NK cells which in return secrets the chemokines such as CXCL10, CXCL12, etc.10 Increased level of CXCL10 and CXCL12 can recruit the neutrophils and T cells respectively, which can initiate and assist in immunopathological mechanisms in lupus patients.11 Activation of immune cells by interleukins and recruitment of immune cells by chemokines leads to the formation of a pathogenic loop which can be one of the causes in lupus complexity. Elevation in MIF level was known to reverse the anti-inflammatory effect of glucocorticoids, while the increased in SCF level might enhance the proliferation of myeloid cells having importance in lupus pathology. Thus, the obtained data pointed towards the interplay of pro-inflammatory cytokine and chemokines which can modulates the SLE pathogenesis.

Interestingly, the levels of cytokines such as MIP-1b/CCL4, IL-9, 1L-18, IL-12 and PDGF was increased in more severe patients with SLEDAI score >12. CCL4 was demonstrated in recruitment of monocytes and T cells and IL-12 and IL-18 can induce the Th1 response which facilitates the type I response including secretion of IL-9.12 PDGF can recruit the mast cells and IL-9 triggers the secretions from mast cells to accelerate the immune response.13 Therefore, the interplay of elevated cytokines among severe patients shows the importance of type 1 immune response in exacerbating the lupus towards more severity.

Protein interaction analysis shows that the majority of significantly altered cytokines in HC vs SLE are functionally interconnected and multiple interaction among the CXCL1, CXCL10, CCL5, CXCL12, and CXCL9, constitute a core complex. Functional interconnection between core complex showed their close interplay in accelerating the immune response as CCL5 known to facilitates the macrophage-NK migration and T cell/DC interaction and CXCL1, CXCL9, CXCL10, and CXCL12 regulates the Th1 response, Th1, CD8, neutrophils and NK trafficking which can potentially modulates the immunobiology of SLE.14 Majority of these chemokines involved in T and B cell induced local and renal inflammation in lupus.15 Interaction of the core complex with the others cytokines such as IL-10, IL-4, CSF3R, CCL7, CCL2 and CCL27 demonstrates the induction of anti-inflammatory in response to inflammatory milieu through IL-4 and IL-10 secretion.16 However, no significantly difference was observed among IL-4 and IL-10 level on comparison among HC vs SLE group. CCL7 and CCL2 also contributes in local and renal inflammation in lupus nephritis.17 Taken together, these data indicate the impaired functional interactions among chemokines and interleukins enhanced the dysregulated immune response in SLE.

Correlation analysis among SLE showed the densely related cytokines when compared with healthy control's correlation matrix. LIF, FGF, and IP-10/CXCL10 were majorly involved in correlation as the central modulator in SLE. LIF and CXCL10 can potentially modulate the SLE manifestations including the lupus nephritis and was positively correlated with ANA level. Increased FGF level in lupus patients was found to be associated with neuropsychiatric lupus conditions. Negative correlation among Gro-a∼FGF∼IP-10/CXCL10 and b-NGF∼IL-10 demonstrated the response of anti-inflammatory cytokines such as IL-10 and b-NGF to elevated level of Gro-a/CXCL1 and CXCL10.18 ROC analysis showed the possibilities of these chemokines and cytokines (AUC value of >0.9) such as Gro-a/CXCL1, SDF-1a/CXCL12, M-CSF, MIF, and IL-7, IL-18 as potential biomarkers for SLE. However, further studies are required with more number of patients for better efficacy of these biomarkers.

Furthermore, the cytokine profiles in HC vs RA showed the increased level of various cytokines in RA patients such as the MIP-1b/CCL4, PDGF, IL-9, IL-4, TNF-b, IP-10/CXCL10, RANTES/CCL5, SDF-1/CXCL12, HGF, MIP, TNF-a, and CTACK/CCL27 and these observations were similar to the previous literature.19 Ability of CCL4 in recruiting the monocytes and T cells which can secrete TNF-a and IL-4, an important factor for B cell survival.20 But an impaired B cell response may also trigger the autoimmune response in RA which can exacerbates the disease condition. High plasma level of IL-9, CXCL10 and CXCL12 can also recruit the neutrophils at joints which can further tigger the inflammation by secreting NETs along with various inflammatory cytokines among RA patients.21 The cytokines that were observed to be lower among RA patients included IL-15, G-CSF, MCP-3/CCL7, MCP-1/CCL2, IL-10, and Gro-a/CXCL1. As majority of RA patients were on methotrexate dosing, that might be the reason for the reduced level of these chemokines.

PPI analysis among significantly altered cytokines from RA shows the core complex of the chemokines such as the CXCL10, CCL5, CXCL12, CXCL9, CXCL1 and CXCL27, which was similar to the SLE PPI analysis. Involvement of these chemokines as the central modulator among these two pathologies demonstrated its importance as potential biomarker as well as the targets for the various therapeutics. The interplay of these chemokines in RA pathology might be of importance as CXCL10/12, and CCL5 can recruit the immune cells at joints or in synovium of the RA patients and can also regulate the Th1 immune response in RA by facilitating the NETs secretions from the neutrophils.22 The interactions of the core complex with other cytokines such as IL-7, IL-18, CSF3, and CCL7 demonstrated their synergistic interplay as IL-7 is secreted from the inflamed tissue which can further activate the synovial fibroblasts to secret the GM-CSF, facilitating the recruitment of different granulocytes.23 IL-18 was demonstrated to be positively correlated with TNF-a and IL-1b level in synovial fluid which can harsh the disease condition.24

Correlation analysis among RA patients’ cytokines were plotted which showed the interaction among few cytokines when compared with SLE. IL-18, RANTES/CCL5, and G-CSF emerges out as the central connector. Majority of these were chemokines which can recruit the various immune cells contributing to the high inflammatory milieu in RA.25 Further analysis was performed to explore their importance as potential biomarker where the RANTES, TNF-b, IL-4, MIP-1b, G-CSF and IL-9 shows the AUC of 1. However, more extensive studies are required with large number of samples to confirm these findings.

SLE and RA involves the interplay of similar kind of cytokines which makes it difficult to distinguished between them and majority of the lupus patients experienced arthritis at least once during the active disease. Therefore, the comparison of cytokine profile was performed among both the diseases i.e., SLE vs RA, which shows the differentially altered cytokines from which majority of the cytokines were following the same pattern as of HC vs SLE/RA. The commonly altered cytokines among SLE and RA includes IL-9, Gro-a/CXCL1, RANTES/CCL5, IP-10, TNF-b, and MCP-3/CCL7. We have reported few common cytokines following the similar trends among both the pathologies such as SDF-1a/CXCL12 and IP-10/CXCL10, which were increased while MCP-3/CCL7 and Gro-a/CXCL1 were decreased. Rest of the common cytokines was considered to be specific for disease conditions as IL-9, RANETS/CCL5 and TNF-b was decreased among SLE patients while increased in RA. PPI interaction among common significantly altered cytokines in SLE vs RA shows a complex core which was similar to HC vs SLE/RA core which established the importance of CXCL1, CXCL10, and CCL5 amid both the pathologies. These cytokines have prominent AUC values therefore possess the potential to be utilized as biomarkers.

Further comparison was done among lupus patients on the basis of the presence or absence of arthritis. The significant alteration in cytokines shows the increased level of cytokines in patients with arthritis which includes IL-12, SCF, and TNF-a and decreased level of eotaxin/CCL11, IL-9, RANTES, and TNF-b. Generally, the level of all these cytokines is observed to be elevated among both the diseases,26 but the difference among with/without arthritis condition might be of clinical interest. Eotaxin/CCL11 can also be utilized as biomarker in lupus to discriminate between the non-nephritis vs nephritis patients.27 Moreover, a major fact remains that the alteration in the cytokines level can also be altered due to the prescribed treatment among the patients. The level of TNF-a was increased among the RA patients and lupus patients with arthritis while decreased in SLE patients including without arthritis conditions. This result shows the TNF-a as a potential indicator for arthritis condition. However, the level of TNF-a was also demonstrated high among the lupus patients.28 PPI interactions among common cytokines in lupus with arthritis vs without arthritis shows the multifunctional interactions of CCL5, CCL11, TNF-a and TNF-b (LTA). This core complex was different from all previous PPI interactions which might be specific for lupus with arthritis condition, however, CCL5 was observed as common player among all the PPI analysis and have good AUC value which further endorse its potential role among these disorders. CCL5 shows positive correlation with oxidative stress and can induce tissue damage in SLE and RA29 and was previously also proven as biomarker in RA which fans the possibility for CCL5 to be considered as a biomarker for autoimmune response.

The current scenario of therapy for most autoimmune condition includes therapeutics that target the symptomatic results of the disease and thus providing momentary relief but require a lifetime of medications and thorough management of the disease. Moreover, the autoimmune conditions have ability to induce other autoimmune conditions, thus we require some common targets which can be targeted in multiple manifestations. These potential biomarkers may be utilised to for prognostic purposes and can also act as futuristic therapeutic targets to alleviate the effects of disease pathogenesis in various autoimmune conditions such as SLE and RA. To further explore the possible therapeutics to target the potential cytokines that emerge during the different comparisons in the current study include CXCL1, CCL5, CXCL10, CXCL12, CCL7, and IL-9, top 10 predicted drugs were screened out from DSigDB database and among these drugs, acetovanillone (CTD00002374) observed to be a potential drug due its least p value and have targets for majority of aforesaid cytokines. Acetovanillone is a plant-based ketone which has been previously used to treat many disorders such as cardio and hepatotoxicity,30 however its role in targeting of SLE and RA is still undercover.

Limitations of any scientific study are important and help in building a better future study. The limitation of the current study is sample size; therefore, the obtained results should be inferred with caution. Another factor to be borne in mind is to remember that the patients are on treatment which might influence the cytokines levels. Although the cytokines interplay can be of similar nature among majority of autoimmune disorders, however, their implication as biomarkers requires more extensive studies. The outcomes of this study draw a conclusion where the differential interplay of cytokines varies among both disorders, SLE and RA, which can provide a diagnostic approach to differentiate among both the pathologies based on the cytokines level. In addition to this, the common cytokines which emerges out from various comparison pointed towards the similar mechanisms involved in both diseases which can be utilised as targets as well as prognostic paradigm for their detection. SLE patients can include arthritis as one of the important comorbidities which makes the disease more severe and these patients observed different cytokine alterations from non-arthritic ones, which can also assist in planning of the therapeutic regime. Hence, the present study gives important pointers towards the relevance of using various cytokine profiles as biomarkers for differentiating the two autoimmune disorders. At the same time, therapeutic targets based on differences between the two disorders can be closely followed for futuristic studies.

AA, RB, and JK contributed to analysing of the data, designing of the study, and writing the manuscript. AB and AS provided the critical contributions and reviewing the manuscript.

Human ethical permission was approved by post graduate institute of medical education and research (PGIMER), Chandigarh (IEC# IEC-05/2016-419 and IEC-01/2022-2277).

We acknowledge the University Grant Commission (UGC) for fellowship to Mr. Akhil. Financial assistance received from Department of Biotechnology (DBT)-BUILDER project to carry out this work are also acknowledged.

The authors declare no conflict of interest.