IL-23, but not IL-12, plays a critical role in inflammation-mediated bone disorders.

Interleukin-12 (IL-12) and IL-23 are thought to have central roles in inflammation and are critical to pathologies associated with inflammation-induced bone disorders. The deletion of IL-12p40 (a common subunit of IL-12 and IL-23) can improve bone regeneration. However, the relative roles of IL-12 and IL-23 in bone disorders are largely unknown. Methods: Ectopic bone formation and skull defect models were established to evaluate the relative roles of IL-12 and IL-23 in inflammatory bone disorders. Differences in bone mass among WT, IL-12p35-/-, and IL-12p40-/- mice (young and elderly) were detected by micro-CT. Osteogenic and osteoclastic activities were explored using ELISA, qRT-PCR, and histological analysis. Moreover, the mechanisms by which IL-12 and IL-23 regulated the differentiation of BMMSCs and RAW264.7 cells were explored using Alizarin Red and tartrate-resistant acid phosphatase staining in vitro. Apilimod was used to inhibit IL-12 and IL-23 production in vivo. Results: Mice deficient in IL-12p40 promoted bone formation and protected against aging-related bone loss. By contrast, bone loss was aggravated in IL-12-/- mice, suggesting that IL-23 may play a dominant role in inflammation-related bone disorders. Mechanistically, IL-12 and IL-23 coupled osteogenesis and osteoclastic activities to regulate bone homeostasis and repair. IL-23 deficiency increased bone formation and inhibited bone resorption. Finally, apilimod treatment significantly improved bone regeneration and calvarial defect repair. Conclusion: These data collectively uncover a previously unrecognized role of IL-23 in skeletal tissue engineering. Thus, IL-23 can act as a biomarker to predict diseases and treatment efficacy, and apilimod can be used as an effective therapeutic drug to combat inflammatory bone disorders.

IL-35: a potential target for the treatment of atherosclerosis.

The imbalance of anti- inflammatory/pro-inflammatory cytokines plays an important role in the process of atherosclerosis. IL-35 is an anti-inflammatory cytokine comprising the p35 subunit of IL-12 and the subunit Epstein-Barr virus (EBV) -induced gene 3(EBI3). Accumulating evidence showed that IL-35 up-regulates the expression of anti-inflammatory cytokines, induces the generation of CD4 + regulatory T cells, inhibits CD4 + effector T cells response and other target cells activity, and reduces the progression of inflammatory and autoimmune diseases. In addition, it has been found that Ebi3 and p35 strongly coexpressed in human advanced lesions. Therefore, we hypothesize that IL-35 may become a novel target for the treatment of atherosclerosis. Further studies are required to investigate the precise effect and the signaling pathway of IL-35 in atherosclerosis process.

Interleukin-12p35 deletion promotes CD4 T-cell-dependent macrophage differentiation and enhances angiotensin II-Induced cardiac fibrosis.

OBJECTIVE: Interleukin-12 is essential for the differentiation of naïve T cells into interferon-γ-producing T cells, which regulate inflammatory responses. We investigated this process of regulating hypertension-induced cardiac fibrosis. METHODS AND RESULTS: Mice infused with angiotensin II showed a marked increase in interleukin-12p35 expression in cardiac macrophages. The degree of cardiac fibrosis was significantly enhanced in interleukin-12p35 knockout (p35-KO) mice compared with wild-type (WT) littermates in response to angiotensin II. Fibrotic hearts of p35-KO mice showed increased accumulation of alternatively activated (M2) macrophages and expression of M2 genes such as Arg-1 and Fizz1. Bone marrow-derived macrophages from WT or p35-KO mice did not differ in differentiation in response to angiotensin II treatment; however, in the presence of CD4(+) T cells, macrophages from p35-KO mice differentiated into M2 macrophages and showed elevated expression of transforming growth factor-ß. Moreover, CD4(+) T-cell-treated p35-KO macrophages could stimulate cardiac fibroblasts to differentiate into α-smooth muscle actin-positive and collagen I-positive myofibroblasts in 3-dimensional nanofiber gels. Neutralizing antibodies against transforming growth factor-ß inhibited myofibroblast formation induced by M2 macrophages. CONCLUSIONS: Deficiency in interleukin-12p35 regulates angiotensin II-induced cardiac fibrosis by promoting CD4(+) T-cell-dependent differentiation of M2 macrophages and production of transforming growth factor-ß.

Airway inflammation and IgE production induced by dust mite allergen-specific memory/effector Th2 cell line can be effectively attenuated by IL-35.

CD4(+) memory/effector T cells play a central role in orchestrating the rapid and robust immune responses upon re-encounter with specific Ags. However, the immunologic mechanism(s) underlying these responses are still not fully understood. To investigate this, we generated an allergen (major house dust mite allergen, Blo t 5)-specific murine Th2 cell line that secreted IL-4, IL-5, IL-10, and IL-13, but not IL-9 or TNF-α, upon activation by the cognate Ag. These cells also exhibited CD44(high)CD62L(-) and CD127(+) (IL-7Rα(+)) phenotypes, which are characteristics of memory/effector T cells. Experiments involving adoptive transfer of this Th2 cell line in mice, followed by three intranasal challenges with Blo t 5, induced a dexamethasone-sensitive eosinophilic airway inflammation. This was accompanied by elevation of Th2 cytokines and CC- and CXC-motif chemokines, as well as recruitment of lymphocytes and polymorphic mononuclear cells into the lungs. Moreover, Blo t 5-specific IgE was detected 4 d after the last intranasal challenge, whereas elevation of Blo t 5-specific IgG1 was found at week two. Finally, pulmonary delivery of the pVAX-IL-35 DNA construct effectively downregulated Blo t 5-specific allergic airway inflammation, and i.m. injection of pVAX-IL-35 led to long-lasting suppression of circulating Blo t 5-specific and total IgE. This model provides a robust research tool to elucidate the immunopathogenic role of memory/effector Th2 cells in allergic airway inflammation. Our results suggested that IL-35 could be a potential therapeutic target for allergic asthma through its attenuating effects on allergen-specific CD4(+) memory/effector Th2 cell-mediated airway inflammation.

Cytokine responses and inducible nitrous oxide synthase expression patterns in neonatal chicken brain microglia infected with very virulent Marek's disease virus strain YL040920.

Purified and enriched brain microglia from neonatal chickens were infected with live Marek's disease virus (MDV)-both the very virulent (vv) YL040920 strain and the attenuated vaccine strain CVI988/Rispens in vitro. Although YL040920-infected microglia showed lower viral DNA loads compared with those infected with CVI988/Rispens at the same infectious dose (400 plaque-forming units for each), no significant differences in IFN-γ and IL-12p35 transcription were detected between the two MDV strains. Chicken microglia infected with live or fixed YL040920 expressed dramatically higher levels of IL-12p40, IL-8, and macrophage inflammatory protein-1ß (MIP-1ß) transcripts compared with those infected with CVI988/Rispens. On the other hand, CVI988/Rispens induced significantly higher levels of IFN-ß transcription than YL040920, especially the live virus. Inducible nitric oxide (NO) synthase (iNOS) transcription and NO production correlated with levels of both YL040920 and CVI988/Rispens live strain infection. Moreover, fixed MDVs induced higher levels of iNOS/NO than live viruses, especially with CVI988/Rispens. This study demonstrates that chicken microglial cells can become infected with live YL040920 and CVI988/Rispens and that microglia represent cellular sources of IL-12p40, IL-12p35, IFN-γ, IFN-ß, IL-8, MIP-1ß, iNOS mRNA, and NO expression after MDV infection in vitro. Transcription levels of IL-12p35 and IFN-γ were associated with MDV DNA replication, whereas transcription levels of IL-12p40, IFN-ß, IL-8, and MIP-1ß were associated with both MDV DNA replication and expression of viral specific genes. The transcription of iNOS was responsible for expression of viral specific genes, whereas it was suppressed by viral DNA replication during infection. Although YL040920, compared with CVI988/Rispens, induced similar levels of the typical Th1-type cytokine IFN-γ in microglia, vvMDV induced significant increases in other cytokines [IL-12 (p40 and 12p35), IL-8, and MIP-1ß]. More detailed investigation, as well as in vivo testing of the effects of vvMDV infection on Th1 responses, iNOS expression, and NO production in the brain of chickens should be undertaken.

IL-12p35 promotes antibody-induced joint inflammation by activating NKT cells and suppressing TGF-beta.

The functional role of IL-12 in rheumatoid arthritis is controversial. Moreover, whether IL-12 contributes to regulation of Ab-induced joint inflammation remains unclear. To address these issues, we explored the functional roles of IL-12 in Ab-induced arthritis using the K/BxN serum transfer model. IL-12p35(-/-) and IL-12Rbeta(2)(-/-) mice were resistant to the development of arthritis. Injection of K/BxN serum into IL-12p40-yellow fluorescence protein reporter (yet40) mice induced CD11b(+) cells, CD11c(+) cells, and Gr-1(+) granulocytes to produce IL-12p40 in the joints. The levels of IFN-gamma, IL-4, and IL-6 production were lower in joint tissues of IL-12p35(-/-) and IL-12Rbeta(2)(-/-) mice than in B6 mice, whereas levels of TGF-beta expression were higher. Administering IL-12p35(-/-) mice rIL-12 or IFN-gamma restored joint inflammation and suppressed TGF-beta production in joint tissues. Moreover, administering neutralizing anti-TGF-beta mAb enhanced joint inflammation. Among the immune cells that infiltrated joint tissues during Ab-induced arthritis, NKT cells expressed IL-12beta(2) receptors. Furthermore, the adoptive transfer of splenocytes from B6 or Gr-1(+) granulocyte-depleted mice restored joint inflammation in IL-12Rbeta(2)(-/-) mice as much as in B6 mice, whereas splenocytes from Jalpha18(-/-) mice did not. These findings indicate that signals via IL-12beta(2) receptors on NKT cells play a critical role in the development of Ab-induced arthritis. The IL-12p35/IFN-gamma axis promotes Ab-induced joint inflammation by activating NKT cells and suppressing TGF-beta, which may provide novel information for the development of new therapeutic strategies for the inhibition of rheumatoid arthritis.

A novel p40-independent function of IL-12p35 is required for progression and maintenance of herpes stromal keratitis.

PURPOSE. Interleukin (IL)-12p40 can couple with IL-12p35 or p19 chains to form the molecules IL-12p70 and IL-23, respectively, which promote T(H)1 cytokine responses. IL-12p35 can bind to EBI3 to form the anti-inflammatory molecule IL-35, but a proinflammatory function of IL-12p35 independent of IL-12p40 has not been described. Here such a function in a mouse model of herpes stromal keratitis (HSK), a CD4(+) T(H)1 cell-dependent corneal inflammation, is demonstrated. METHODS. Corneas of wild-type (WT), IL-12p40(-/-), IL-12p35(-/-), and IL-12p35(-/-)p40(-/-) (double knockout) mice were infected with the RE strain of HSV-1, and HSK was monitored based on corneal opacity, neovascularization, leukocytic infiltrate, and cytokine/chemokine levels. RESULTS. All mouse strains developed moderate HSK by 11 days after infection (dpi). However, from 11 to 21 dpi, HSK progressed in WT and IL-12p40(-/-) mice but regressed in IL-12p35(-/-) and IL-12p35(-/-)p40(-/-) mice. HSK regression was characterized by reductions in neutrophils and CD4(+) T cells and attenuation of blood vessels, which was associated with reduced levels of the chemokines KC (CXCL3), Mip-2 (CXCL2), and MCP-1 (CCL2) and the angiogenic factor vascular endothelial growth factor. CONCLUSIONS. HSK development does not require IL-12p40 and is thus independent of IL-12p70 and IL-23. However, late HSK progression does require a previously unrecognized IL-12p40-independent, proinflammatory function of IL-12p35.

IL-12 produced by dendritic cells augments CD8+ T cell activation through the production of the chemokines CCL1 and CCL17.

IL-12 family members are an important link between innate and adaptive immunity. IL-12 drives Th1 responses by augmenting IFN-gamma production, which is key for clearance of intracellular pathogens. IL-23 promotes the development of IL-17-producing CD4(+) T cells that participate in the control of extracellular pathogens and the induction of autoimmunity. However, recent studies have shown that these cytokines can modulate lymphocyte migration and cellular interactions. Therefore, we sought to determine the individual roles of IL-12 and IL-23 in naive CD8(+) T cell activation by addressing their ability to influence IFN-gamma production and cellular interaction dynamics during priming by Listeria monocytogenes-infected dendritic cells (DC). We found that IL-12 was the major cytokine influencing the level of IFN-gamma production by CD8(+) T cells while IL-23 had little effect on this response. In addition, we observed that IL-12 promoted longer duration conjugation events between CD8(+) T cells and DC. This enhanced cognate interaction time correlated with increased production of the chemokines CCL1 and CCL17 by WT but not IL-12-deficient DC. Neutralization of both chemokines resulted in reduced interaction time and IFN-gamma production, demonstrating their importance in priming naive CD8(+) T cells. Our study demonstrates a novel mechanism through which IL-12 augments naive CD8(+) T cell activation by facilitating chemokine production, thus promoting more stable cognate interactions during priming.