Irina Kochetkova

IL-35 Stimulation of CD39+ Regulatory T Cells Confers Protection against Collagen II-Induced Arthritis via the Production of IL-10

IL-35 is produced by regulatory T cells, and this novel cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. In this work, an rIL-35, as a single-chain fusion between murine IL-12p35 and EBV-induced gene 3, was expressed in yeast. This rIL-35 inhibited OVA-specific cellular and Ab responses in OVA-challenged recipients of DO11.10 CD4+ T cells. Likewise, IL-35 inhibited clinical manifestation of collagen-induced arthritis or could cease further disease exacerbation upon initiation of IL-35 treatment. Exogenous IL-35 treatments suppressed Th1 and Th17 cells and promoted CD39 expression by CD4+ T cells. Sorted CD25−CD39+CD4+ T cells from IL-35–treated mice produced IL-10 and, upon adoptive transfer, were sufficiently potent to inhibit subsequent development of inflammation in mice with collagen-induced arthritis, whereas sorted CD25+CD39+CD4+ T cells showed reduced potency. IL-35 treatments of IL-10−/− mice failed to induce protective CD39+CD4+ T cells, demonstrating the effector role of IL-10 by IL-35 immunosuppression.

IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen.

Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25+CD4+ regulatory T (Treg) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact Treg cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/IIC). SJL mice were challenged with proteolipid protein peptide 139–151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/IIC greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN-γ but diminished TGF-β production by Treg cells from Salmonella-CFA/IIC-treated mice. Adoptive transfer of Treg cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25−CD4+ T cells from Salmonella-CFA/IIC showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-γ neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/IIC-induced Treg cells. Thus, the Salmonella-CFA/IIC vaccine elicits Treg cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these Treg cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.

Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression

Human IBD, including UC and Crohnˈs disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti‐inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL‐1β, TNF‐α, IL‐6, IL‐17, IL‐22, CXCL9, CXCL10, CXCL11, and IFN‐γ in the colons of mice with colitis. APP‐mediated protection requires T cells, as protection was abated in Rag‐1−/− or TCRα−/− mice but not in IL‐10−/−, IRF‐1−/−, μMT, or TCRδ−/− mice. Administration of APP during colitis to TCRα−/− mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαβ cells in APP‐mediated protection. APP treatment also inhibited CXCR3 expression by TCRαβ cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4+ or CD8+ T cells alone did not abolish APP‐mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.

Vaccination without Autoantigen Protects against Collagen II-Induced Arthritis via Immune Deviation and Regulatory T Cells

Anti-inflammation immunotherapy has been successfully applied for the treatment of autoimmune diseases. Mucosal vaccines against autoimmune disorders are beneficial by influencing the regulatory compartment of gut and systemic adaptive immune systems. A Salmonella vector expressing colonization factor Ag I (CFA/I), shown to behave as an anti-inflammatory vaccine, stimulates the production of CD4+CD25+ T cells and regulatory cytokines. In this work, we queried whether Salmonella-CFA/I can protect DBA/1 mice from collagen-induced arthritis. The incidence of arthritis and cartilage loss in vaccinated DBA/1 mice was remarkably lower when compared with unprotected mice. Clinical findings were accompanied by the suppression of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-27. Vaccination evoked a multi-tier response consisting of IL-4 producing Th2 cells, an increased production of TGF-β by CD4+ T cells, and suppression of collagen II-specific CD4+ T cell proliferation. To assess the contribution of Salmonella-CFA/I-primed CD4+ T cells, adoptive transfer studies with total CD4+, CD4+CD25−, or CD4+CD25+ T cells were performed 15 days postchallenge. Mice receiving either subset showed reduced disease incidence and low clinical scores; however, mice receiving total CD4+ T cells showed delayed disease onset by 10 days with reduced clinical scores, reduced IL-17 and IL-27, but enhanced IL-4, IL-10, IL-13, and TGF-β. Inhibition of TGF-β or IL-4 compromised protective immunity. These data show that Salmonella-CFA/I vaccination of DBA/1 mice protects against collagen-induced arthritis by stimulating TGF-β- and IL-4-producing regulatory CD4+ T cells.

Segregated Regulatory CD39+CD4+ T Cell Function: TGF-β–Producing Foxp3− and IL-10–Producing Foxp3+ Cells Are Interdependent for Protection against Collagen-Induced Arthritis

Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-γ via enhanced IL-4, IL-10, and TGF-β. To identify the responsible regulatory CD4+ T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39+CD4+ T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39+CD4+ T cells into CIA mice conferred complete protection, whereas CD39−CD4+ T cells did not. Subsequent analysis of vaccinated Foxp3-GFP mice revealed the CD39+ T cells were composed of Foxp3-GFP− and Foxp3-GFP+ subpopulations. Although each adoptively transferred Salmonella-CFA/I–induced Foxp3− and Foxp3+CD39+CD4+ T cells could protect against CIA, each subset was not as efficacious as total CD39+CD4+ T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed Foxp3− CD39+CD4+ T cells produced TGF-β, and Foxp3+CD39+CD4+ T cells produced IL-10, showing a segregation of function. Moreover, donor Foxp3-GFP− CD4+ T cells converted to Foxp3-GFP+ CD39+CD4+ T cells in the recipients, showing plasticity of these regulatory T cells. TGF-β was found to be essential for protection because in vivo TGF-β neutralization reversed activation of CREB and reduced the development of CD39+CD4+ T cells. Thus, CD39 apyrase-expressing CD4+ T cells stimulated by Salmonella-CFA/I are composed of TGF-β–producing Foxp3− CD39+CD4+ T cells and support the stimulation of IL-10–producing Foxp3+ CD39+CD4+ T cells.

Identification and characterization of a novel class of c-Jun N-terminal kinase inhibitors.

In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.

IFN-γ-deficient mice develop IL-1-dependent cutaneous and musculoskeletal inflammation during experimental brucellosis

Human brucellosis exhibits diverse pathological manifestations that can affect almost any organ. In particular, osteoarticular complications are the most common focal manifestation of brucellosis and occur in 40–80% of patients. In immunocompetent mice, Brucella replication is generally restricted to the spleen, liver, and to a lesser extent, LNs, thereby limiting their use for study of focal inflammation often found in brucellosis. Here, we report that nasal, oral, or peritoneal infection of IFN‐γ−/− mice with WT Brucella melitensis or Brucella abortus results in joint and periarticular tissue inflammation. Histological analysis of the affected joints revealed inflammatory infiltrates and debris within the joint space colocalizing with Brucella antigen. Osteoarthritis, necrosis, periarticular soft tissue inflammation, and substantial brucellae burdens were observed. Oral rifampicin was effective in clearing infection and halting further progression of focal inflammation from infected IFN‐γ−/− mice, although some symptoms and swelling remained. Elevated IL‐1β, but not TNF‐α, IL‐6, or IL‐17, was detected in joint homogenates from infected IFN‐γ−/− mice. Whereas more susceptible to systemic infection, IL‐1R−/− mice depleted of IFN‐γ were more resistant to focal inflammation than WT mice similarly depleted of IFN‐γ. Collectively, these results show IFN‐γ−/− mice represent a potential model for study of focal inflammation attributed to Brucella infection and will allow evaluation of intervention strategies targeting IL‐1, IL‐1R, or other inflammatory mediators, with the potential to complement antibiotic‐based therapies.

Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor

c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S–treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II–specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II–specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3+CD4+CD25+ regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis.

Oral Escherichia coli Colonization Factor Antigen I Fimbriae Ameliorate Arthritis via IL-35, Not IL-27

A Salmonella therapeutic expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-β–producing Foxp3−CD39+CD4+ T cells and IL-10–producing Foxp3+CD39+CD4+ T cells. However, it is unclear whether CFA/I fimbriae alone are protective and whether other regulatory cytokines are involved, especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as did Salmonella-CFA/I and found that Foxp3+CD39+CD4+ T cells were the source of secreted IL-35, whereas IL-27 production by CD11c+ cells was inhibited. Inquiring into their relevance, CFA/I fimbriae–treated IL-27R–deficient (WSX-1−/−) mice were equally protected against CIA as were wild-type mice, suggesting a limited role for IL-27. In contrast, CFA/I fimbriae–mediated protection was abated in EBI3−/− mice, accompanied by the loss of TGF-β– and IL-10–producing Tregs. Adoptive transfer of C57BL/6 CD39+CD4+ T cells to EBI3−/− mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory collagen II–specific Th cells. In contrast, recipients cotransferred with C57BL/6 and EBI3−/− CD39+CD4+ T cells and treated with CFA/I plus IL-35 were not protected, implicating the importance of endogenous IL-35 for conferring CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the coinduction of TGF-β and IL-10.

The Absence of Lymphoid CD8+ Dendritic Cell Maturation in L-Selectin−/− Respiratory Compartment Attenuates Antiviral Immunity

Intratracheal instillation of L-selectin-deficient (L-Sel−/−) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or β-galactosidase-specific CTLs with concomitant long-lived β-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c+CD8+ dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel−/− CTL activity, cell-sorted L-Sel−/−CD8+ T cells were cocultured with cell-sorted L-Sel+/+CD8+ or CD8− DCs or L-Sel−/−CD8− DCs. Only the CD8+ DCs restored CTL activity; L-Sel−/−CD8− DCs failed to support L-Sel+/+ CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8+ DCs were detected, the DC environment remained suppressive in L-Sel−/− mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel−/− adoptively transferred with OT-1 CD8+ T cells. To assess whether the L-Sel−/−CD8− DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel−/− LRLN to make matured DCs. When L-Sel−/− mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8+ DCs in L-Sel−/− mice. These studies show that impairment of L-Sel−/− DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8+ DCs to support CTL responses.