In this study, an unbiased investigation was performed to predict biological targets and activities of LMWF5A in an immune cell model. This was accomplished by determining DEG upon LMWF5A treatment of stimulated PBMC followed by in silico analysis with IPA software. Evaluation of graphical summaries generated by IPA revealed that the overall biological trend observed in PBMC stimulated with LPS, LPS/IFNγ, or IL-4/IL-13 in the presence of LMWF5A compared to saline control is the suppression of immune cell inflammatory activities, as numerous key pro-inflammatory regulators and functions were predicted to be inhibited and some anti-inflammatory regulators were predicted to be activated with LMWF5A. These predictions support the potential use of LMWF5A in a range of clinical conditions that result from a hyperactive or chronic immune response.
To delineate the predicted effects of LMWF5A on immune cells more specifically, known molecules with similar or opposite activities to LMWF5A were identified via upstream regulator analysis in IPA. The most similar upstream regulators were DEX, SIRT1, PTGER4, filgrastim, and SB203580. The glucocorticoid DEX, as a synthetic steroid, has multiple indications, including inflammatory conditions (such as rheumatoid and psoriatic arthritis, systemic lupus erythematosus, and Crohn’s disease), multiple sclerosis, cerebral edema, shock, and allergies, amongst others . Importantly, DEX has also been administered as an intra-articular injection for joint inflammation  and has been recently studied as a potent anti-inflammatory for the treatment of COVID-19 ; both conditions are currently being investigated in LMWF5A clinical trials.
SIRT1 is an NAD-dependent protein deacetylase that negatively regulates inflammation by altering cytokine levels and immune cell recruitment and activation by deacetylating and suppressing transcription factors, including NFκB . Similarly, LMWF5A has also been proven to modulate transcription factor activity to decrease pro-inflammatory cytokine levels. Its ability to reduce TNF and IL-1β release has been linked to its effects on the NFκB-repressing PPARγ and AhR pathways in LPS-stimulated PBMC, and it has also been shown to prevent NFκB reporter activity .
PTGER4 (or EP4) is a transmembrane, G-coupled protein receptor that becomes activated when bound to the cyclooxygenase (COX) pathway product PGE2, which is induced during inflammation. Although the role of PGE2 is pleotropic, it exerts its anti-inflammatory effects via PTGER4. PTGER4 binds to EP4 receptor-associated protein, which in turn, reduces the phosphorylation and increases the stability of p105, an inhibitor of NFκB and mitogen-activated protein/extracellular signal-regulated kinase (MEK) . Overall, PTGER4 downregulates inflammation by modulating macrophage cytokine and chemokine secretion as well as T cell proliferation, differentiation, and cytokine production . LMWF5A has been associated with COX pathway upregulation and its products, including the PTGER4 ligand PGE2, in PBMC and primary human osteoarthritic cells [5, 10]. The mode of action of LMWF5A may be unique to the anti-inflammatory drug class, as most inhibit both cytokine and prostaglandin release while LMWF5A inhibits cytokine release but promotes prostaglandin release. Inhibition of the COX/prostaglandin pathway can result in harmful side effects , and stimulation of this pathway with concomitant inhibition of cytokine production by LMWF5A may, conversely, promote anti-inflammation, resolution, and healing.
Filgrastim, recombinant human granulocyte colony stimulating factor, is commonly used clinically as a complement to chemotherapy due to its ability to stimulate granulocyte production, thus preventing low white blood cell counts . In addition, this protein has confirmed anti-inflammatory properties in vivo with respect to the cytokine response and has been suggested as a potential treatment for chronic inflammatory conditions; for example, LPS-induced cytokine release has been shown to be attenuated in healthy human volunteers treated with filgrastim [43, 44].
Finally, SB203580 is a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, with strong effects on cytokine production . The p38 MAPK signaling pathway is critical to the regulation of many cellular processes, including inflammation, as it is activated in response to inflammatory mediators and other stress-related molecules and acts as a major regulator of cytokine production . Overall, the top upstream regulators that significantly match LMWF5A activity have been empirically proven to downregulate components critical to inflammation, including pro-inflammatory cytokines and transcription factors.
The identification of upstream regulators with opposing activity to LMWF5A revealed LPS, poly rI:rC-RNA, IFNγ, IFNα2, and STAT1 as the molecules with the most disparate effects. All of these molecules are known promoters of inflammation, as recognition of PAMPs and DAMPs by pattern recognition receptors, like TLRs, results in signal transduction to turn on multiple transcription factors (NF-κB, MAPK, STAT) that increase expression of key pro-inflammatory cytokines .
LPS and poly rI:rC-RNA are both categorized as PAMPs and function as signals of infection to initiate immune signaling via TLRs. LPS is a major component of Gram-negative bacterial cell walls, and acts as a PAMP upon bacterial infection. It is widely utilized in vitro to stimulate the TLR4-induced immune response that can occur upon recognition of either pathogens or endogenous molecules released upon tissue damage . LPS was used in two of the three conditions in this study to stimulate an immune response in PBMC, and its ranking as the most negatively correlated upstream regulator to LMWF5A highlights the fact that LMWF5A strongly counteracts TLR4-mediated inflammation. Moreover, the anti-inflammatory effects of LMWF5A on cytokine release and transcription factor activity have been extensively studied using cells treated with LPS as a TLR4 stimulant [7, 9, 48]. poly rI:rC-RNA is a dsRNA mimic that can simulate infection with a dsRNA virus and activate TLR3 , suggesting that LMWF5A may also offset the actions of other TLR-driven pathways; the relationships between LMWF5A and other TLRs are currently under investigation.
As additional opposing upstream regulators, the cytokines IFNγ and IFNα2 represent both classes of interferons, type II and I, respectively. They are secreted upon viral infection to limit viral replication and regulate the subsequent immune response . The Janus kinase (JAK)-STAT signaling pathway is the most studied IFN-related transcription factor pathway, but IFNs also activate other signaling cascades, including p38 MAPK and phosphatidylinositol 3'-kinase, to exert their anti-viral and pro-inflammatory effects .
Because both type I and II IFNs activate STAT complexes, it is not surprising that STAT1 is also part of this list of opposing upstream regulators. Upon recognition of IFNs, a variety of interleukins, or other cytokines, STAT1 is phosphorylated, mainly by JAK kinases, and activated to drive a pro-inflammatory cascade . LMWF5A was also previously demonstrated to inhibit the ability of STAT1 to bind its cognate DNA sequence in LPS-stimulated PBMC, implicating regulation of this transcription factor as part of the LMWF5A mode of action . Inhibition of the JAK-STAT pathway is an effective therapeutic strategy for the treatment of inflammatory conditions, including rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease  as well as COVID-19 . In summary, the analysis of inversely correlated upstream regulators of LMWF5A represent pro-inflammatory factors, emphasizing the anti-inflammatory activity of LMWF5A.
Comparison of the common targets between DEX and LMWF5A highlighted that a large proportion, one-third to one-half depending on the stimulation conditions, of the genes regulated by LMWF5A are also regulated by DEX. While most of the genes were regulated in a similar direction to DEX, others were directionally inconsistent.
An example of a target that is regulated by LMWF5A in a direction consistent with DEX is C–C motif chemokine ligand 2 (CCL2), also called monocyte chemoattractant-1. Upon recognition of inflammatory stimuli, CCL2 expression is induced, and this chemokine drives migration of immune cells, particularly monocytes, to the site of infection or tissue injury . Dysregulated, increased CCL2 expression is linked to the pathology of many diseases, including heart failure, RA, and diabetes, due its overpromotion of immune cell infiltration and downstream pro-inflammatory effects . Serum CCL2 has been demonstrated to be increased in OA patients versus healthy controls, suggesting its importance to OA pathogenesis . Some studies have reported increased CCL2 levels in OA synovial fluid as well, and CCL2 has been linked to OA-associated pain in addition to factors influencing cartilage catabolism . With respect to COVID-19, CCL2, along with many other inflammatory cytokines, contributes to the cytokine storm that occurs during the body’s dysregulated response to SARS-CoV2 infection and its level has been correlated with increased disease severity . Thus, the similar activities of both DEX and LMWF5A on CCL2 should provide benefit to patients with CCL2-related diseases, including OA and COVID-19.
Conversely, an example of a target that is regulated by LMWF5A in a direction inconsistent with DEX is cathepsin B (CTSB). CTSB is member of the cathepsin family of cysteine proteases, which are localized in the lysosome . It is well studied in the context of cancer  but has also been implicated in cartilage degradation and OA pathogenesis due to its proteolytic activity of extracellular matrix components and its ability to promote the activity of other proteases . In fact, the enzymatic activity of CTSB was demonstrated to be enhanced in vitro in stimulated primary chondrocytes and in vivo in cartilage, serum, and synovial fluid from OA patients, in which CTSB activity levels were associated with OA disease severity . In the IPA upstream regulator analysis, the IPA Knowledge Database records CTSB as being upregulated by DEX, supporting the cartilage-degrading effects observed with corticosteroids, while the differential expression analysis showed that CTSB was downregulated by LMWF5A. This and other differences may be important distinctions between these two anti-inflammatory treatments. Data comparing LMWF5A directly to DEX in PBMC have been published for a small number of targets. These data showed that in LPS-stimulated PBMC, DEX inhibited TNF, PGE2, and 15d-PGJ2, whereas LMWF5A inhibited TNF but increased levels of PGE2 and 15d-PGJ2 by ELISA . While it is possible to explore each specific directionally consistent or inconsistent target, the present analysis allows for a broad understanding of overall LMWF5A activity and its comparison to DEX as well as the interplay between common targets and their downstream pathways.
The differences in common targets between these datasets points to a limitation of the current study. This analysis solely examined the effects of LMWF5A in one cell model at one time point, albeit with three immunostimulation conditions. Additional timepoints or cell types would provide even further insight into the mode of action of LMWF5A and may identify more overlapping targets. For example, due to the known effects of LMWF5A on cytokine release from PBMC, an earlier timepoint may have captured more differentially expressed cytokine RNAs as well as any temporal differences in cytokine RNA levels due to LMWF5A treatment.
Although all molecules identified in these analyses shed light on the putative effects of LMWF5A, the distinction that DEX was scored by IPA as the most similar upstream regulator prompted further analysis of the LMWF5A datasets with public datasets of DEGs in stimulated PBMC treated with DEX. Of the top 32 upstream regulators as identified in the comparison analysis between DEX and LMWF5A datasets and ranked by absolute z-score, only 11 had > 2 LMWF5A datasets with a non-significant z-score compared to the DEX datasets (Fig. 5). In addition, the observed z-scores and adjusted p-values were not as strongly significant for each of the LMWF5A datasets compared to the DEX datasets, supporting the hypothesis that while LMWF5A acts similarly to DEX, it is not identical to DEX.
Extensive information on the MOA of LMWF5A has been gained with the datasets in this investigation, allowing for the formation of multiple hypotheses that can be tested in future experiments. For instance, targeted experiments exploring the effects of LMWF5A will be performed using DEX as a comparative treatment. These experiments will focus on components of various signaling pathways, including IFN-mediated signaling due to the high instance of IFN-related factors that were predicted to be affected by LMWF5A, including IRFs, STAT1, and both type I and II IFNs themselves. In addition, several specific, inflammatory disease-associated, directionally consistent, and inconsistent targets of LMWF5A and DEX will be investigated, for example CCL2 and CTSB as mentioned above.