Signaling pathways underlying TGF-ß mediated suppression of IL-12A gene expression in monocytes.

Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine essential for multiple biological processes, including the regulation of inflammatory and immune responses. One of the important functions of TGF-ß is the suppression of the proinflammatory cytokine interleukin-12 (IL-12), which is crucial for mounting an anti-tumorigenic response. Although the regulation of the IL-12p40 subunit (encoded by the IL-12B gene) of IL-12 has been extensively investigated, the knowledge of IL-12p35 (encoded by IL-12A gene) subunit regulation is relatively limited. This study investigates the molecular regulation of IL-12A by TGF-ß-activated signaling pathways in THP-1 monocytes. Our study identifies a complex regulation of IL-12A gene expression by TGF-ß, which involves multiple cellular signaling pathways, such as Smad2/3, NF-κB, p38 and JNK1/2. Pharmacological inhibition of NF-κB signaling decreased IL-12A expression, while blocking the Smad2/3 signaling pathway by overexpression of Smad7 and inhibiting JNK1/2 signaling with a pharmacological inhibitor, SP600125, increased its expression. The elucidated signaling pathways that regulate IL-12A gene expression potentially provide new therapeutic targets to increase IL-12 levels in the tumor microenvironment.

Immunomodulatory effect of probiotic exopolysaccharides in a porcine in vitro co-culture model mimicking the intestinal environment on ETEC infection.

The aim of this study was to evaluate the immunomodulatory effect of EPS-L26 isolated from the probiotic strain Lactobacillus (Limosilactobacillus) reuteri L26 Biocenol™, in a model of infection with an enterotoxigenic E. coli (ETEC) by establishing monocultures consisting of the IPEC-J2 cell line or monocyte-derived dendritic cells (moDCs) and creating a 3D model of cell co-cultures established with IPEC-J2 cells and moDCs. The immunomodulatory and immunoprotective potential of used EPS-L26 was confirmed in monocultures in an experimental group of pretreated cells, where our study showed that pretreatment of cells with EPS-L26 and subsequent exposure to infection resulted in significantly down-regulated mRNA levels of genes encoding inflammatory cytokines compared to ETEC challenge in single cell cultures (in IPEC-J2, decreased mRNA levels for TNF-α, IL-6, IL-1ß, IL-12p35; in moDCs, decreased mRNA levels for IL-1ß). Similar to monocultures, we also demonstrated the immunostimulatory potential of the ETEC strain in the co-culture model on directly treated IPEC-J2 cells cultivated on insert chambers (apical compartment) and also on indirectly treated moDCs cultivated in the lower chamber (basolateral compartment), however in the co-culture model the expression of inflammatory cytokines was attenuated at the mRNA level compared to monocultures. Pretreatment of the cells on the insert chambers pointed to the immunoprotective properties of EPS-L26, manifested by decreased mRNA levels in both cell lines compared to ETEC challenge (in IPEC-J2 decreased mRNA levels for IL-12p35; in moDCs decreased mRNA levels for IL-1ß, IL-6). Our results suggest intercellular communication via humoral signals derived from IPEC-J2 cells by influencing the gene expression of indirectly treated moDC cells located in the basolateral compartment.

Teprotumumab Divergently Alters Fibrocyte Gene Expression: Implications for Thyroid-associated Ophthalmopathy.

CONTEXT: Teprotumumab, an IGF-I receptor (IGF-IR) inhibitor, is effective in thyroid-associated ophthalmopathy (TAO). The drug can modulate induction by TSH of IL-6 and IL-8 in CD34+ fibrocytes and their putative derivatives, CD34+ orbital fibroblasts (CD34+ OF). Fibrocytes express multiple thyroid autoantigens and cytokines implicated in TAO, which are downregulated by Slit2. Inflammation and disordered hyaluronan (HA) accumulation occur in TAO. Whether teprotumumab alters these processes directly in fibrocytes/CD34+ OF remains uncertain. OBJECTIVE: Determine teprotumumab effects on expression/synthesis of several TAO-relevant molecules in fibrocytes and GD-OF. DESIGN/SETTING/PARTICIPANTS: Patients with TAO and healthy donors were recruited from an academic endocrine and oculoplastic practice. MAIN OUTCOME MEASURES: Real-time PCR, specific immunoassays. RESULTS: Teprotumumab attenuates basal and TSH-inducible autoimmune regulator protein, thyroglobulin, sodium iodide symporter, thyroperoxidase, IL-10, and B-cell activating factor levels in fibrocytes. It downregulates IL-23p19 expression/induction while enhancing IL-12p35, intracellular and secreted IL-1 receptor antagonists, and Slit2. These effects are mirrored by linsitinib. HA production is marginally enhanced by teprotumumab, the consequence of enhanced HAS2 expression. CONCLUSION: Teprotumumab affects specific gene expression in fibrocytes and GD-OF in a target-specific, nonmonolithic manner, whereas IGF-IR control of these cells appears complex. The current results suggest that the drug may act on cytokine expression and HA production systemically and locally, within the TAO orbit. These findings extend our insights into the mechanisms through which IGF-IR inhibition might elicit clinical responses in TAO, including a potential role of Slit2 in attenuating inflammation and tissue remodeling.

Mucosa-Associated Invariant T Cell Hypersensitivity to Staphylococcus aureus Leukocidin ED and Its Modulation by Activation.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib-related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57+ NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

The subunits of IL-12, originating from two distinct cells, can functionally synergize to protect against pathogen dissemination in vivo.

Cytokines are typically single gene products, except for the heterodimeric interleukin (IL)-12 family. The two subunits (IL-12p40 and IL-12p35) of the prototype IL-12 are known to be simultaneously co-expressed in activated myeloid cells, which secrete the fully active heterodimer to promote interferon (IFN)γ production in innate and adaptive cells. We find that chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, generate functional IL-12. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism is sufficient to propel local T cell differentiation in sites distal to the initial infection and helps control systemic dissemination of a pathogen, although not parasite burden, at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival.

Local Interleukin-12 Treatment Enhances the Efficacy of Radiation Therapy by Overcoming Radiation-Induced Immune Suppression.

Radiation therapy (RT) recruits myeloid cells, leading to an immunosuppressive microenvironment that impedes its efficacy against tumors. Combination of immunotherapy with RT is a potential approach to reversing the immunosuppressive condition and enhancing tumor control after RT. This study aimed to assess the effects of local interleukin-12 (IL-12) therapy on improving the efficacy of RT in a murine prostate cancer model. Combined treatment effectively shrunk the radioresistant tumors by inducing a T helper-1 immune response and influx of CD8+ T cells. It also delayed the radiation-induced vascular damage accompanied by increased α-smooth muscle actin-positive pericyte coverage and blood perfusion. Moreover, RT significantly reduced the IL-12-induced levels of alanine aminotransferase in blood. However, it did not further improve the IL-12-induced anti-tumor effect on distant tumors. Upregulated expression of T-cell exhaustion-associated genes was found in tumors treated with IL-12 only and combined treatment, suggesting that T-cell exhaustion is potentially correlated with tumor relapse in combined treatment. In conclusion, this study illustrated that combination of radiation and local IL-12 therapy enhanced the host immune response and promoted vascular maturation and function. Furthermore, combination treatment was associated with less systemic toxicity than IL-12 alone, providing a potential option for tumor therapy in clinical settings.

Silencing IL12p35 Promotes Angiotensin II-Mediated Abdominal Aortic Aneurysm through Activating the STAT4 Pathway.

Background and Purpose. Abdominal aortic aneurysm (AAA) is a chronic inflammatory disorder and the important causes of death among men over the age of 65 years. Interleukin-12p35 (IL12p35) is an inflammatory cytokine that participates in a variety of inflammatory diseases. However, the role of IL12p35 in the formation and development of AAA is still unknown. Experimental Approach. Male apolipoprotein E-deficient (Apoe-/-) mice were generated and infused with 1.44 mg/kg angiotensin II (Ang II) per day. We found that IL12p35 expression was noticeably increased in the murine AAA aorta and isolated aortic smooth muscle cells (SMCs) after Ang II stimulation. IL12p35 silencing promoted Ang II-induced AAA formation and rupture in Apoe-/- mice. IL12p35 silencing markedly increased the expression of inflammatory cytokines, including IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α), in both the serum and AAA aorta. Additionally, IL12p35 silencing exacerbated SMC apoptosis in Apoe-/- mice after Ang II infusion. IL12p35 silencing significantly increased signal transducer and activator of transcription (STAT) 4 phosphorylation levels in AAA mice, and STAT4 knockdown abolished the IL12p35-mediated proinflammatory response and SMC apoptosis. Interpretation. Silencing IL12p35 promotes AAA formation by activating the STAT4 pathway, and IL12p35 may serve as a novel and promising therapeutic target for AAA treatment.

Importance of particle size of oligomannose-coated liposomes for induction of Th1 immunity.

Oligomannose-coated liposomes (OMLs) comprised of dipalmitoylphosphatidylcholine, cholesterol and Man3-DPPE at a molar ratio of 1:1:0.1 and particle diameters of about 1000 nm can induce liposome-encased antigen-specific strong Th1 immunity. In this study, we evaluated the effect of particle sizes of OMLs on induction of Th1 immune responses in mice. Spleen cells obtained from mice immunized with antigen-encapsulating OMLs with 1000- and 800-nm diameters secreted remarkably high levels of IFN-γ upon in vitro stimulation. In addition, sera of mice that received these OMLs had significantly higher titers of antigen-specific IgG2a than those of IgG1, which are commonly associated with Th1 responses. In contrast, treatment with antigen-encapsulating OMLs with 400- and 200-nm diameters failed to induce IFN-γ secretion from spleen cells, although these OMLs did elicit elevation of antigen-specific IgGs. In addition, the titers of serum antigen-specific IgG2a were the same as those of IgG1 in mice that received 400-nm OMLs. Resident peritoneal mononuclear phagocytes (MNPs) treated with OMLs of diameter ≥ 600 nm secreted IL-12, which is essential for induction of Th1 immune responses, while those treated with OMLs of ≤ 400 nm failed to produce this cytokine. However, 400-nm OMLs did induce enhanced expression of MHC class II and costimulatory molecules on MNPs, similarly to OMLs of ≥ 600 nm. Taken together, these results strongly indicate that OMLs of diameter ≥ 600 nm are required to induce Th1 immune responses against OML-encased antigens, although OMLs of diameter ≤ 400 nm can activate MNPs.

The characterization of an interleukin-12 p35 homolog involved in the immune modulation of oyster Crassostrea gigas.

Vertebrate interleukin-12 (IL-12) is a heterodimeric cytokine composing of two subunits (p35 and p40). In the present study, a p35-like subunit homolog of vertebrate IL-12 was identified from oyster Crassostrea gigas (designated as CgIL12p35L), with an open reading frame of 411 bp encoding a putative peptide of 136 amino acids. There was a long four-helix chain in CgIL12p35L, which was similar as that in vertebrate IL-12 p35. Comparative genomic analysis showed that there were conservative kinds of syntenic genes flanked CgIL12p35L. The mRNA transcripts of CgIL12p35L were constitutively expressed in various tissues and its mRNA expression level in haemocytes increased significantly after bacteria challenge. The activity of haemolymph to eliminate bacteria from the oysters treated with recombinant CgIL12p35L protein (rCgIL12p35L) in vivo increased significantly. The results collectively indicated that the homolog of vertebrate IL-12 p35 subunit existed in oysters, and it was involved in immune defense against bacteria challenge.

Neutrophil functions can be regulated by IL-35, which is mainly expressed in IL-15Rα+ cells in grass carp (Ctenopharyngodon idella).

IL-35 plays a key role in regulatory T (Treg) and regulatory B (Breg) cell functions in mammals. CD25 has been demonstrated as one of the markers of Treg cells, and CD19+CD25hiCD71hi cells have been verified as a type of Breg cells in humans. These results indicate that there is a close relationship between IL-35 and CD25+ cells. In mammals, CD25 (alias IL-2Rα) has been identified as having high affinity and specificity for IL-2 binding, and is closely linked and structurally related to IL-15Rα, which having high affinity for IL-15 binding. In teleost, IL-15Rα can bind to both IL-2 and IL-15, with higher affinity to IL-15 than IL-2, and has been termed a CD25-like molecule in some research studies. To date, no studies of IL-35 and IL-15Rα have been documented in fish. In this work, five isoforms of IL-15Rα were cloned from grass carp, and a monoclonal antibody to the protein was developed. The results of flow cytometry and quantitative real-time PCR analyses demonstrated that grass carp IL-35 subunit genes EBI3a and IL-12p35 were mainly expressed in IL-15Rα+ cells, while the expression levels of IL-10 and TGF-ß in IL-15Rα+ and IL-15Rα- cells were insignificant. Recombinant grass carp IL-35 (rgcIL-35) could increase the proportion of IL-15Rα+ cells in leukocytes, and a certain proportion of IL-15Rα+ cells also appeared in myeloid cell subset II after stimulation with rgcIL-35. Meanwhile, the migration, phagocytic ability, and bactericidal ability of grass carp neutrophils were significantly decreased after stimulation with certain concentrations of rgcIL-35. Moreover, neutrophil apoptosis could be significantly inhibited by rgcIL-35.

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

Retinoid Regulation of Ocular Surface Innate Inflammation.

Corneal and conjunctival inflammation and dry eye develop in systemic vitamin A deficiency (VAD). The objective of this study was to investigate the lacrimal ocular surface retinoid axis, particularly immunomodulatory effects of retinoic acid (RA) and change in conjunctival myeloid cell number and phenotype in VAD. We discovered that ocular surface epithelial and myeloid cells express retinoid receptors. Both all trans- and 9-cis-RA suppressed production of dry eye relevant inflammatory mediators [interleukin(IL)-1ß, IL-12, regulated upon activation, normal T cell expressed and secreted (RANTES)] by myeloid cells. Systemic VAD was associated with significant goblet cell loss and an increased number of CD45+ immune cells in the conjunctiva. MHCII-CD11b+ classical monocytes were significantly increased in the conjunctiva of VAD C57BL/6 and RXR-α mutated Pinkie strains. RNA seq revealed significantly increased expression of innate immune/inflammatory genes in the Pinkie conjunctiva. These findings indicate that retinoids are essential for maintaining a healthy, well-lubricated ocular surface and have immunomodulatory effects in the conjunctiva that are mediated in part via RXR-α signaling. Perturbation of the homeostatic retinoid axis could potentiate inflammation on the ocular surface.

Interleukin-35 regulates the balance of Th17 and Treg responses during the pathogenesis of connective tissue diseases.

Interleukin (IL)-35 belongs to the IL-12 cytokine family and is a heterodimer of the p35 and Epstein-Barr virus-induced gene 3 (EBI3) subunits. Functionally, IL-35 can promote the proliferation and activation of regulatory T cells (Tregs) and suppress the function of T helper 17 (Th17) cells and other inflammatory cells to inhibit immune responses. In recent years, an abnormal IL-35 expression causing a Th17/Treg imbalance has been associated with the development and progression of several connective tissue diseases (CTDs), such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), dermatomyositis (DM)/polymyositis (PM), and primary Sjögren's syndrome (pSS). Here, we review the role of IL-35 in regulating the balance of Th17/Treg responses in different types of CTDs and provide new insights into the role of IL-35 in these diseases.

FXTAS presents with upregulation of the cytokines IL12 and TNFα.

INTRODUCTION: Fragile X Tremor and Ataxia Syndrome is a progressive neurodegenerative disorder that develops in some FMR1 premutation carriers. The objective of this study is to characterize how cytokine levels are altered in the FXTAS brain. METHODS: Fresh frozen cerebellar tissue from FXTAS cases and controls was homogenized and analyzed for 12 different cytokines using a commercially available ELISA panel. RESULTS: Relative to controls, FXTAS cases showed large and significant increases in the cytokines IL-12 and TNFα. There were large but non-significant increases in the levels of IL-2, IL-8, and IL-10 in FXTAS cases. The cytokines IL-1A, IL-1B, IL-4 IL-6, IL-17A, IFNγ, and GM-CSF were not different between FXTAS and control subjects. CONCLUSIONS: For the first time, we demonstrate an increase in the pro-inflammatory cytokines TNFα and IL-12 in the FXTAS brain, both of which are implicated in the pathogenesis of Multiple Sclerosis, another neurodegenerative disorder that predominantly consists of white matter disease.

Pyruvate Kinase M2 Promotes the Activation of Dendritic Cells by Enhancing IL-12p35 Expression.

Dendritic cells (DCs) play a central role in both innate and adaptive immunity. Emerging evidence has demonstrated metabolic reprogramming during DC activation. However, how DC activation is linked with metabolic reprogramming remains unclear. Here we show that pyruvate kinase M2 (PKM2), the rate-limiting enzyme in the last step of glycolysis, is critical for LPS-induced DC activation. Upon DC activation, JNK signaling stimulated p300 association with PKM2 for the acetylation of lysine 433, a classic posttranslational modification critical for PKM2 destabilization and nuclear re-localization. Subsequently, nuclear PKM2 partnered with c-Rel to enhance Il12p35 expression, which is important for Th1 cell differentiation. Meanwhile, decreased enzymatic activity of PKM2 due to detetramerization facilitated glycolysis and fatty acid synthesis, helping DCs meet their need for biomacromolecules. Together, we provide evidence for metabolic control of DC activation and offer insights into aberrant immune responses due to dysregulated Th1 functions.

IL-35 promotes microglial M2 polarization in a rat model of diabetic neuropathic pain.

Switching microglial polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype represents a novel therapeutic strategy for diabetic neuropathic pain (DNP). This study aims to determine the role and mechanism of interleukin (IL)-35 in regulating microglial M1/M2 polarization in DNP. A rat model of DNP was induced by a single streptozocin injection and recombinant IL-35 (rIL-35) was then intrathecally administered to the rats for 14 days. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to assess the therapeutic effect of IL-35. Highly aggressive proliferating immortalized (HAPI), a rat microglia cell line, was treated with lipopolysaccharide (LPS) for M1 polarization or IL-4 for M2 polarization. The M1 markers (CD68, iNOS, TNF-α, IL-6) and M2 markers (CD206, Arg-1, IL-10) were examined. rIL-35 administration in DNP model rats elevated MWT and TWL, induced microglial polarization toward the M2 phenotype, suppressed JNK signaling and activated JAK2/STAT6 signaling. In vitro assay confirmed that rIL-35 induced microglial M2 polarization in HAPI cells through inhibiting JNK signaling and activating JAK2/STAT6 signaling. Collectively, the mechanism underlying therapeutic effect of IL-35 on DNP may relate to its promotion of microglial M2 polarization by regulating JNK signaling and JAK2/STAT6 signaling.

Dynamic changes of innate lymphoid cells in acute ST-segment elevation myocardial infarction and its association with clinical outcomes.

An increasing body of evidence has implicated the innate immune system in the causation of acute ST-segment elevation myocardial infarction (STEMI). Innate lymphoid cells (ILCs) are newly identified members of the lymphoid lineage that are important effectors of innate immunity. The role of ILCs in STEMI has not been explored. We characterized the ILCs present in peripheral blood of 176 STEMI patients and 52 controls. Patients were followed up for up to 23 months. Flow cytometry showed that the proportion of total ILCs and ILC1s were significantly increased compared with controls; contrary to ILC1s, the proportion of ILC2s among total ILCs decreased significantly during the acute phase of STEMI. ILC1s percentage was an independent predictor of major adverse cardiovascular events (MACE). On multivariate Cox regression, the 3rd tertile of ILC1s was associated with a higher MACE rate compared with the 1st tertile (hazard ratio: 2.26; 95% confidence interval 1.56-3.27; P = 0.014). RNA-sequencing (RNA-Seq) revealed increased expressions of interferon-γ, tumor necrosis factor-α, vascular cell adhesion molecule 1 (VCAM1), and matrix metallopeptidase 9. Moreover, as active factors secreted by ILC1s, levels of interleukin (IL)-12 and IL-18 were significantly increased in STEMI patients. Increased ILC1s in patients with STEMI was associated with poor outcomes. Our findings suggest that ILC1s may play an important role in STEMI.

Treg-Cell-Derived IL-35-Coated Extracellular Vesicles Promote Infectious Tolerance.

Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in TregEbi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3+ and Foxp3neg (iTr35) T cells. Furthermore, IL-35 producers, although rare, secrete Ebi3 and p35 on extracellular vesicles (EVs) targeting a 25- to 100-fold higher number of T and B lymphocytes, causing them to acquire surface IL-35. This surface IL-35 is absent when EV production is inhibited or if Ebi3 is genetically deleted in Treg cells. The unique ability of EVs to coat bystander lymphocytes with IL-35, promoting exhaustion in, and secondary suppression by, non-Treg cells identifies a novel mechanism of infectious tolerance.

Serum level of IL-4 predicts response to topical immunotherapy with diphenylcyclopropenone in alopecia areata.

BACKGROUND: This study investigated predictors of response to topical diphenylyclopropenone (DPCP) immunotherapy in patients with alopecia areata (AA). OBJECTIVE: To identify predictors of response, or resistance, to treatment for AA through clinical observations and serum tests. METHODS: Eighty four AA patients were treated with DPCP. Serum cytokine levels were measured in 33 AA patients pre- and post-treatment, and in 18 healthy controls, using ELISA assays. RESULTS: Of patients, 56.1% responded to DPCP with satisfactory hair regrowth; the response rate was negatively correlated with hair loss extent. Before DPCP treatment, higher serum IFN-γ and IL-12 cytokine levels were observed in AA patients compared to healthy controls. Non-responders to DPCP had significantly elevated serum IL-4 pre-treatment (3.07 fold higher) and lower IL-12 levels compared with responders. After DPCP treatment, non-responders had persistently high IL-4, increased IL-12, negligible decrease in IFN-γ and decreased IL-10. Post-treatment DPCP responders exhibited significantly decreased IFN-γ and IL-12, and increased IL-4 and IL-10. Development of adverse side-effects was significantly associated with higher pre-treatment serum IgE levels. LIMITATIONS: A small number of subjects were evaluated. CONCLUSIONS: Potentially, elevated pre-treatment serum levels of IL-4 and IL-12 can be used as unfavorable and favorable predictors of DPCP therapeutic effect, respectively. In addition, pre-treatment elevated serum total IgE may predict increased risk for severe adverse side-effects to DPCP application. Whether serum cytokine expression levels can be used as predictors of response to other forms of treatment is unknown, but it may warrant investigation in the development of personalized treatments for AA.

Pancreatic cancer-associated inflammation drives dynamic regulation of p35 and Ebi3.

B cells are important modulators of immune responses both in autoimmunity and cancer. We have previously shown that B regulatory (Breg) cells promote pancreatic cancer via production of IL35, a heterodimeric cytokine comprised of the subunits p35 (Il12a) and Ebi3. However, it is not known how production of IL35 is regulated in vivo in the context of cancer-associated inflammation. To begin addressing this question, we have generated a knock-in mouse model, Il12aGFP, where an IRES-emGFP gene was inserted within the 3' UTR of the Il12a locus. EmGFP signal in B cells from the Il12aGFP mice correlated with expression of p35 mRNA and protein. Using this model, we observed that in addition to Bregs, expression of GFP (p35) is upregulated in several other B cell subtypes in response to cancer. We assessed the expression of the other IL35 subunit, Ebi3, using a published tdTomato reporter model. We determined that Ebi3 expression was more tightly regulated in vivo and in vitro, suggesting that stimuli affecting Ebi3 upregulation are more likely to result in production of full IL35 heterodimer. We were also able to detect GFP and Tomato signal in myeloid & T cell lineages suggesting that these reporter models could also be used for tracking IL12-, IL27- and IL35-producing cells. Furthermore, using primary B cells isolated from reporter mice, we identified BCR, CD40 and TLR pathways as potential drivers of IL35 expression. These findings highlight the importance of pancreatic cancer-associated inflammatory processes as drivers of cytokine expression and provide a tool to dissect both disease-associated regulation of IL12- and IL35-competent lineage cells as well as establish assays for pharmacological targeting of individual subunits of heterodimeric IL12 family cytokines.