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Probiotic bacteria are live organisms, if consumed in adequate amounts might confer health benefits. These bacteria, such as Lactic acid bacteria (LAB), include a number of strains that have specific health promoting activi-ties, attributed to their immunomodulatory and anti-inflammatory properties. Gut mucosal macrophage subsets play a fundamental role in driving muco-sal immune responses. These include, tolerance, associated with an M2-, regulatory macrophage phenotype and inflammatory activation with an M1-like phenotype. The cross-link between mucosal tolerance and inflammatory cytokine suppression, and augmentation of IL-10 production in the gut relate to endotoxin tolerance. Endotoxin tolerance is a context; it could present an example for cell drive through a hypo-responsive state. An example is mu-cosal inflammatory pathologies, such as Crohn's disease. When tolerance is broken, causing the destruction of gut mucosal tissue. This is where the macrophage phenotype, has been transformed from a regulatory M2- to an inflammatory M1-like phenotype. This is seen as a reaction to both, patho-genic and commensal bacteria. This investigation was aimed at assessing the activities of live probiotic bacteria; Lactobacillus salivarius strain MS13 and Lactobacillus plantarum strain C28 in the immunomodulation of macro-phage subsets in health, inflammation, and endotoxin tolerance. M1- and M2-like macrophages were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and Vitamin D3, respectively. Additionally, differentiated epithelial cells (Caco-2) were obtained by long term culturing for 21 days. The role of Lactobacillus strains C28 and MS13 to modulate epi-thelial barrier integrity and macrophage-epithelial cell inflammation was in-vestigated. TNFα, IL-1β, IL-18, IL-23, IL-12, IL-6, IL-8, and IL-10 were quanti-fied by ELISA and RT-PCR, whereas TLR-2, TLR-4, Tollip, SOCS3, STAT3 and TRAIL by RT-PCR. This study revealed that, first, live C28 and MS13 stimulated the proinflammatory cytokine by M2-like macrophages as well as the anti-inflammatory cytokine in a homeostatic status; whereas in an in-flammatory environment, C28 and MS13 differentially upregulated TNFα and IL-1β by M1 and M2-like macrophages induced by E.coli K12-LPS. Both strains downregulated K12-LPS induced IL-10 by M2-like macrophages. The response of stimulated M1 and M2 macrophages to C28 and MS13, was to differentially induce the gene expression of TLR-2, TLR-4, Tollip, NLRP3, SOCS-3, STAT3 and TRAIL. Second, the repeat-stimulation/tolerisation of M1 and M2 macrophages by live probiotic bacteria revealed, TNFα, IL-1β, IL-23, IL-18, IL-6 and IL-10 were upregulated in M1-like macrophages by C28, whereas MS13 upregulated TNFα, IL-1β, IL-18, and downregulated IL-12, IL-6, and IL-10. On the other hand, the tolerisation of M2-like macrophages by C28 and MS13 resulted in the downregulation of TNFα and IL-12p35 and upregulation of IL-1β, IL-18, IL-23, IL-12, IL-6, and IL-10. These findings were linked with the differential macrophage subset upregulation of TLR-4, NLRP3, STAT-3 and TRAIL gene expression. On the other hand, TLR-2, Tol-lip and SOCS-3 were downregulated in tolerised macrophage subsets by C28 and MS13. Furthermore, the role of lactobacilli strains C28 and MS13 in the modulation of endotoxin tolerance was to; upregulate TNF-α, IL-18, IL-23 and IL-10 by M1 and M2-like macrophages. This investigation also focused on the induction of the zona-occludin-1 (Zo-1), human β defensin-2 (hBD-2), and cytokine production IL-8 by Caco-2 cells. Trans epithelial electrical re-sistance (TEER) and RT-PCR measured the main cytokines studied pro-duced by Caco-2, were IL-8, also the epithelial barrier function. Live probiotic C28 and MS13 suppressed the production of IL-8 (in the presence or ab-sence TNFα and IL-1β). Moreover, in the co-culture of Caco-2 with macro-phage subsets, MS13 enhanced the expression of hBD-2 and ZO-1. These findings allow for the better understanding of live probiotic roles on macro-phage subsets functions and endotoxin tolerisation mechanisms, which may be beneficial for the development of in vivo models of probiotic bacteria and therapeutic targeting of inflammatory bowel disease.

Survival and persistence of live matter requires certain flexibility to allow multiple adaptations, yet it is grounded on orderly and stringent regulation of its important functions. An astounding example of natural planning is given by mammalian embryological development where a sophisticated specification program enclosed in a single cell is resolved into a diversity of tissues in a mature organism. Tracing back the developmental timeline is a way to deconvolute advanced biological properties that cells acquire through a series of hierarchical differentiation decisions. One of the primary unspecified states spanning a short period between conception and functional commitment of the cell is known as pluripotency. Pluripotency is the ability of a cell to give rise to the derivatives of the three germ layers and germ cells during differentiation. Naturally, pluripotency is restricted to the early embryo only and represented by two morphologically, physiologically and epigenetically sequential stages - naïve (embryonic stem cells, or ESCs) and primed (post-implantation epiblast stem cells, or EpiSCc). Revolutionary technologies of nuclear reprogramming are now able to artificially revert developmental vector and direct differentiated cells towards naïve pluripotency. Reprogrammed cells not only come as a useful object for fundamental research but also as a flexible biological tool for regenerative therapies. Currently reprogramming is moderately efficient, however reliable and safe medical strategies require decent understanding of the physiological and genetic networks underpinning cell identity change. Importantly, the primed pluripotent cell can be reprogrammed too allowing to focus on the first and often subtle cell identity transitions that cells adopt. In my project, I have performed a forward genome-wide CRISPR-gRNA knockout screen to identify genes working as barriers for reprogramming from EpiSCs to ESCs. Having assembled a comprehensive lentiviral library and established a tractable cell system to target the mouse genome, I screened EpiSCs in the reprogramming setup which allowed selection of effective reprogramming antagonists. Here I discover the JAK/STAT3 pathway regulator Socs3 as a key screen hit and describe this gene as a self-sufficient and powerful roadblock between the pluripotent identities. I show how a spectrum of its attenuated activities displayed in naïve pluripotent and reprogramming cells may be functionally linked to reinforced pluripotency. Exploring the interplay between JAK/STAT3 pathway and other major naïve pluripotency genes across naïve, primed and non-pluripotent cell contexts reveals partially conservative mode of action of SOCS3. I demonstrate how SOCS3 may integrate external signalling with endogenous core pluripotency factors and by thereby feed in the naïve circuity supporting it and instructing its establishment. This work helps understand the mechanisms behind reprogramming and biology of pluripotency which potentially can provide insights into consolidated and robust medical cell technologies.

Cardiomyocytes and macrophages have been found to interact via connexin-43 hemichannels. The role of connexin-43, however, is not fully understood. This study shows that these interactions aid in increasing the membrane potential of cardiomyocytes allowing contraction of the cells. HL-1 cardiomyocytes and RAW 264.7 macrophages in coculture increased expression of connexin-43 compared to cardiomyocytes alone. Co-cultures also increased the fluorescence of Di-8-ANEPPS potentiometric dye indicating an increase in cardiomyocyte membrane potential. Treatment with IL-10 and TGF-beta further increased connexin-43 expression and membrane potential. Treatment with SOCS3 inhibited the effects of TGF-beta and IL-10 while having no effect on its own. Confocal imaging can provide visual evidence of these interactions with image layering, 3D imaging and enhanced color.

Cadmium (Cd) is a ubiquitous pollutant in the environment and a known carcinogen for lung cancer. Cd has been shown to act as a weak mutagen, which suggests that it may exert tumorigenic effect through non-genotoxic ways, such as epigenetic mechanisms. The goal of this project is to investigate the mechanisms of Cd carcinogenesis focusing on the role of lncRNA dysregulations. The Cd-exposed cells formed significantly more colonies in soft agar, displayed cancer stem cell (CSC)-like property and formed tumors in nude mice. Mechanistically, the lncRNA microarray analysis revealed that chronic Cd exposure dysregulates lncRNA expressions. Q-PCR analysis confirmed the significant upregulation of the oncogenic lncRNA DUXAP10 level in Cd-transformed cells. Knockdown of DUXAP10 in Cd-transformed cells significantly reduced their CSC-like property. Further mechanistic studies showed that DUXAP10 activates the Hedgehog pathway to promote Cd-induced CSC-like property. Furthermore, it was determined that chronic Cd exposure upregulates DUXAP10 expression by inducing Pax6 expression. In addition to oncogenic lncRNA upregulation, Cd exposure was also found to downregulate the expression of a tumor suppressive lncRNA MEG3 in Cd-transformed cells. Meanwhile, the levels of DNMTs in Cd-transformed cells were found significantly elevated. Bisulfite-sequencing study revealed that the differentially methylated region (DMR) upstream of MEG3 is hypermethylated in Cd-transformed cells, indicating that the promoted DNMTs activity contributed to downregulation of MEG3. Stably expressing MEG3 in Cd-transformed cells decreased cell proliferation and induced CSC-like property. Further studies showed that MEG3 inhibits cell transformation by limiting cell proliferation and inducing apoptosis. Mechanistic studies revealed that MEG3 reduced cell proliferation by regulating the levels of cell cycle proteins and induced apoptosis by inhibiting the level of Bcl-xL. These findings suggest that dysregulations of lncRNAs play important roles in Cd carcinogenesis. As well as epigenetic dysregulations, we also determined the effect of genetic factor contributing to lung cancer. Enhanced EGFR signaling contributes to 60% of NSCLC cases. However, there is an unmet need to solve acquired resistance to tyrosine kinase inhibitors and low response rate for immunotherapy in lung cancer patients. This study was performed to investigate the role of SOCS3 in EGFR mutation-driven lung cancer and to explore the potential of its regulatory axis as therapeutic target for the development of novel approach. In our transgenic mouse model, overexpression of SOCS3 significantly inhibited tumor formation with mutated EGFR. Further investigation for the underlying mechanism revealed that SOCS3 downregulates YAP protein, which further suppressed Bcl-2 family proteins. External YAP inhibitor was shown to efficiently inhibit the growth of tumor organoids. In vivo studies demonstrated that SOCS3 downregulating YAP leads to less immunosuppressive tumor microenvironment. Lastly, SOCS3 was often found to be silenced in cancers. To mimic this circumstance, the therapeutic efficacy of utilizing external YAP inhibitor combined with anti-PD-L1 was assessed and showed promising outcomes. These results suggest the critical role of SOCS3 as a biomarker for the oncolytic immune environment and provide a novel insight for improving lung cancer immunotherapy.

Macrophages are phagocytic cells located in tissues, organs and even circulated within our body as white blood cells. They are critical in detecting tissue damage and infection. Resident tissue macrophages initiate the signals for inflammation recruiting neutrophils and blood monocytes which mature into macrophages at sites of infection and in the resolution of inflammation. Based on the local cytokine milieu in tissue sites, macrophages may be polarized into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Receptor tyrosine kinase Mer (MERTK) helps in clearing dead neutrophils and other apoptotic cells from damaged tissue sites preventing chronic inflammation and autoimmune disorders. MERTK aids in the maintenance of tissue homeostasis and wound healing. Phosphatidylserine (PtdSer) present on the surface of apoptotic cells release “eat me” signals which are recognized by the two “bridging ligands” of MERTK receptor, Gas6 and ProS. The binding of the ligands to PtdSer initiates intracellular signals leading to phagocytosis of the cell. MERTK receptor is expressed mostly on M2c macrophages.The current study explores the expression rate of the phagocytic receptor MERTK, on macrophages polarized with either IL-10 (M2c ells) or IL-4 or IL-13 (M2a macrophages) following treatment with the suppressor of cytokine signaling SOCS3 in comparison with macrophage polarization with only IL-10 or IL-4 or IL-13 .The current study exhibits an enhancement in the expression of the phagocytic MERTK receptor on the surface of IL-10 polarized M2c macrophage when treated with SOCS3 in comparison to IL-10 polarized M2c macrophage, IL-4 polarized M2a macrophage and IL-13 treated M2a macrophage. IL-13 polarized M2a macrophage also shows an increase in the expression of MERTK receptor which is similar to a previous study where a similar receptor to MERTK termed “Axl receptor” is enhanced by IL-13 treatment on bone- marrow derived macrophage. SOCS3 when treated with IL-13 polarized M2a macrophage acts as a negative regulator of MERTK receptor by decreasing the expression contrasting to the effect of SOCS3 on IL-10 which enhances the expression.Based on these observations, future research will involve co-culturing SOCS3 polarized M2 macrophages with apoptotic cells such as N2a neuroblastoma cells.

Macrophages are essential phagocytic cells involved in both innate and adaptive immune systems and play vital roles in the host defense and inflammation. Macrophages have a remarkably high capacity to clear unnecessary cellular materials in interstitial environment through a process called “phagocytosis”, which is affected by many factors including suppressors of cytokine signaling (SOCS). SOCSs are a group of intracellular proteins that downregulate the cytokine signals involved in various JAK/STAT pathways through a negative feedback loop. This study focuses on investigating the effects of SOCS1 and SOCS3 on the phagocytic ability of RAW 264.7 macrophages polarized into M2a with IL-4/IL-13 and into M2c with IL-10 to clear fluorescently marked Neuro-2a malignant cells. Calreticulin (CRT) is one of the cellular signals, exposed on the surface of malignant cells, that play vital roles in controlling the phagocytosis process mediate by phagocytes including macrophages. Treatment of M2a (IL-4/IL-13) and M2c macrophages with SOCS1 peptide mimetics had no significant effects on M2a and M2c-mediated phagocytosis of CFSE-stained N2a cells compared with M2a (IL-4/IL-13) and M2c cells without SOCS1 peptide mimetic treatment, while it slightly increased the M2a (IL-4/IL-13), but not M2c, macrophage-mediated phagocytosis of N2a cells that had their calreticulin receptors blocked. When N2a cells were blocked with anti-CRT antibody, M2a and M2c-mediated phagocytosis was decreased. Interestingly, treatment of M2c with SOCS3 peptide mimetics coincided with blockage of CRT signals on N2a cells caused a significant increase in the phagocytosis of N2a cells mediated by M2c polarized macrophages. These data help to get a deeper understanding of macrophage functions in cleaning the microenvironment from apoptotic and malignant cells and even wound healing. Overall, this study sheds more light on how SOCS1/ SOCS3 can be used along with manipulation of “eat-me” signals exposed on malignant and apoptotic cells to increase the clearance rate of malignant cells and decrease the tumor survival.

Mitogen activated protein kinases (MAPKs) are Ser/Thr kinases that relay the extracellular signal into intracellular responses and regulate several biological responses. They are classified into conventional MAPKs and atypical MAPKs. Extracellular signal regulated kinase 3 (ERK3) is an atypical MAPK that has a single phospho-acceptor site (Ser 189) in its activation motif instead of the canonical Thr-Xaa-Tyr (TXY) motif of conventional MAPK like ERK1/2. ERK3 comprises of a unique C terminal tail and a central C34 domain that further distinguishes it from ERK1/2. Moreover, compared to ERK1/2, much less is known about the upstream activators and the downstream targets of ERK3. Here, our study identifies IL-6 signaling to be negatively regulated by ERK3. We show that ERK3 downregulates the IL-6 target genes and STAT3 phosphorylation. IL-6 is a multifunctional pleiotropic cytokine that signals predominantly via JAK/STAT signaling pathway. In response to IL-6, STAT3 is phosphorylated at Tyrosine 705 (Y705) residue by JAK2, which successively activates STAT3 as a transcription factor. The IL-6/STAT3 pathway is negatively regulated by a feedback inhibitor SOCS3 (Suppressor of Cytokine Signaling 3) which binds to JAK2, hence, preventing the phosphorylation and activation of STAT3. Interestingly, this study reveals that ERK3 interacts with SOCS3 via its C34 domain. Until now, there has been no information about the physiological or the biochemical function of the C34 domain of ERK3. Hence this study has identified a novel role of ERK3’s C34 domain. Furthermore, we show that ERK3 facilitates the interaction between SOCS3 and JAK2, promoting the inhibition of STAT3 Y705 phosphorylation and activation. Taken together, the findings of our study provide important and novel insights into the negative regulation of IL-6/STAT3 signaling pathway by ERK3.

HSV-1 causes a life-long infection in its host and has evolved multiple strategies to facilitate infection and evade the immune response. This virus has been found to enter cells by both endocytosis and fusion. The way the virus exploits endocytosis is not fully understood. Recent studies have uncovered roles of Rab GTPases, key regulators in intracellular membrane trafficking pathways, in distinct steps of the HSV-1 life cycle (Raza et al., 2018). This study will focus on analyzing the levels of the late endosomal regulator Rab7 expression in macrophages infected with HSV-1. Revealing the effect of virus on the levels of the Rab7 expression-directed vesicular trafficking pathway in endocytosis will allow insight into possible future therapies. Peptide mimetics of suppressor of cytokine signaling molecules (SOCS1 and SOCS3) were examined for the potential ability to modulate expression of Rab7. Immunofluorescence was used to determine Rab7 expression and F-actin polymerization levels in uninfected and HSV-1 infected unpolarized M0 Raw264.7 murine macrophages and polarized subsets. M1 macrophages were polarized by treatment with lipopolysaccharide (LPS) and interferon gamma (IFN-¿). M2 cells were polarized by treatment with either IL-4 [M2(IL-4)] or IL-10 [M2(IL-10)]. Rab7 expression levels increased over a 24 hour observation period in M0 and M2 cell types independent of virus infection. During this same observation period Rab7 expression levels were significantly decreased in M1 polarized macrophages with HSV-1-infection. Treatment of the various unpolarized and polarized, infected and uninfected groups of RAW264.7 cells with either SOCS1 or SOC3 peptide mimetic caused a significant decrease in Rab7 expression over the 24 hour observation time. During this same time, HSV-1 infection effected a significant decrease in F-actin polymerization levels in virus infected M0 and in both M2 macrophage phenotypes. SOCS1 and SOCS3 peptide mimetics caused an additional decrease in F-actin polymerization in both unpolarized HSV-1-infected M0 cells and in uninfected IL-4 polarized M2 cells. These results indicate that macrophage polarization and SOCS1 and SOCS3 peptide mimetics impact Rab7 expression and F-actin polymerization levels more significantly than HSV-1 does in unpolarized M0 and polarized M1 and M2 RAW264.7 murine macrophages.

Suppressor of Cytokine Signaling (SOCS) proteins are inhibitors of cytokine signaling that function via the Janus kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway. Our lab has previously demonstrated that SOCS3-deficient mouse embryonic fibroblasts (MEFs) display enhanced sensitivity to DNA damaging agents, but the molecular mechanism of this effect is not well understood. In this study, we have confirmed SOCS3 as a radioprotector in fibroblasts and human solid tumors and identified a novel mechanism through which SOCS3 exerts its radioprotective effect. SOCS3 regulates the DNA damage response by promoting p21 induction and controlling STAT3-mediated proliferation. We have found that the transcriptional repressor BCL6 is targeted by SOCS3/STAT3 and this novel axis is responsible for specific control of the G1-S transition, as SOCS3-deficient cells display normal S-phase checkpoint. Further, by overexpressing SOCS3 in human pancreatic cancer cell lines that signal through insulin-like growth factor (IGF) receptor and the RON receptor tyrosine kinase, we have determined that the radioprotective effect of SOCS3 supercedes its ability to block specific kinase pathways that increase radiation resistance in tumor cells.In addition to these novel effects of SOCS3 on the DNA damage response, we have also identified radiation-independent functions for SOCS3 in aggressive, metastatic solid tumors. Initial cDNA microarray studies in SOCS3 knockout MEFs revealed that the pro-inflammatory C-X-C family chemokines CXCL1 and CXCL2 are underrepresented in SOCS3-deficient fibroblasts, due to transcriptional repression by uncontrolled STAT3 signaling. On the basis of these observations, we have identified three molecular subtypes of human pancreatic ductal adenocarcinoma (PDA) based on SOCS3 expression and CXCL1/2 dependence. We have stratified human PDA cell lines into three groups in which SOCS3 is either highly expressed, partially repressed, or severely repressed/totally silenced in response to IL-6 or oncostatin M (OSM). Repression of the SOCS3 gene in PDA is not CpG island methylation-dependent, suggesting a possible novel mechanism through which SOCS3 expression is silenced in PDA. Our laboratory has shown that ectopic expression of SOCS3 can promote CXCL1 gene activation and tumor aggressiveness in the molecular subtype with partial SOCS3 repression but not the others. Thus, the three molecular subtypes of PDA that we have identified display distinct biological behaviors, responses to SOCS3 overexpression and CXCL1-dependence. Using an orthotopic xenograft model of human PDA in immunodeficient mice, we demonstrated the in vivo relevance of the STAT3/SOCS3/CXCL1 axis in tumor aggressiveness and metastatic behavior. Our results suggest a novel mechanism for controlling pro-inflammatory signaling in human solid tumors and that SOCS3 can regulate both metastasis and radiation sensitivity through distinct pathways. Our ability to stratify human PDA on the basis of SOCS3/STAT3/CXCL1/2 dependence has implications for the development of individualized therapy for cancer patients. By targeting STAT3, SOCS3, as well as CXCL1, we can regulate metastatic behavior in solid tumors, and increase radiation sensitivity of tumors in the clinic.

This study examined the effects of HSV-1 infection and IFN-γ treatment on Neuro-2A cells. HSV-1 induces expression of SOCS1 and SOCS3 in infected cells, inhibiting the ability of these cells to produce the pro inflammatory, antiviral cytokine IFN-γ (Nowoslawski and Benveniste, 2011). SOCS1 and SOCS3 levels were determined in IFN-γ-treated cells, virus-infected cells, and cells that were both IFN-γ-treated and virus-infected. Results were compared with untreated, uninfected control cells. Flow cytometry data analysis showed a slight decrease in SOCS1 and SOCS3 protein levels in cells treated with IFN-γ for 6 hours compared to control cells. A significant decrease in SOCS1 and SOCS 3 levels was found in cells treated with IFN-γ for 18 hours. Up regulation of SOCS1 and SOCS3 expression was established in virus-infected Neuro-2A cells but this increase was not statistically significant when compared to control cells. However, culturing cells with IFN-γ for 6 hours prior to virus infection led to a significant (50%) decrease in SOCS1 and SOCS3expression compared to cells treated with IFN-γ alone. This showed that HSV-1 was not able to overcome the antiviral effects of IFN-γ to up regulate SOCS1 and SOCS3 expression. Cytopathic effects assays were performed to determine cell viability among cells treated with IFN-γ for 18 hours, cells infected with virus for 48 hours, and cells treated with IFN-γ for 18 hours followed by 48 hour HSV-1 infection. Pre-treating cells with IFN-γ for 18 hours prior to infection with HSV-1 (0.1 MOI) yielded a cell viability level similar to that of control cells (untreated/uninfected). This indicates that IFN-γ is providing antiviral protection to most, if not all of the cells. Applying these studies in a human cell line and eventually in an animal model would be necessary to find the efficacy of IFN-γ as a HSV-1 therapeutic.

In our current study we examined the effects of HSV-1 challenge on J774A.1 macrophages polarized to either a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Polarized J774A.1 macrophages were characterized using CD14-CD86 and SOCS1-SOCS3 expression levels. SOCS proteins are a family of proteins that are capable of inhibiting cytokine-signaling pathways. HSV-1 up regulates expression of SOCS1 protein levels in infected cells, inhibiting the ability of infected cells to produce proinflammatory products (Nowoslawski Akhtar and Benveniste, 2011). This study shows that signals within the microenvironment play a greater role in macrophage polarization, and SOCS1-SOCS3 expression levels, than does HSV-1 challenge. M1 macrophages showed morphological changes following polarization, a significant decrease in cell viability, a two-fold increase in the number of CD14+-CD86+ cells, similar levels of SOCS1 expression, and a 11-fold decrease in SOCS3 expression when compared to control cells. M2 macrophages also exhibited morphological changes, a slight decrease in cell viability, a 26.0% decrease in the number of CD14+-CD86+ cells, and SOCS1-SOCS3 expression levels similar to that of control cells. Following HSV-1 challenge (0.1 MOI), the majority of M1 macrophages and M2 macrophages appeared rounded, possibly due to disruption of actin filaments. Virus-infected M1 macrophages showed a slight decrease in cell viability when compared to uninfected M1 macrophages. Additionally, the number of CD14+-CD86+ cells of both M1 and M2 phenotypes decreased. M1 macrophages exhibited a 39.9% decrease, while M2 macrophages exhibited a 13.2% decrease. SOCS1 expression levels remained relatively unchanged in virus-infected M1 macrophages, while SOCS3 expression levels increased by 30.4% at 24 hours after infection. Increase in SOCS3 levels is hypothesized to be a protective response of infected M1 macrophages due to the release of high levels of proinflammatory molecules. Alternatively, the SOCS1-SOCS3 ratio remained relatively unchanged in the anti-inflammatory phenotype.