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.

Deficiency of IL12p40 (Interleukin 12 p40) Promotes Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysm.

Objective- Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40-/- (interleukin 12 p40) mice, we investigated the effects of M2-predominant macrophages on the development of abdominal aortic aneurysm. Approach and Results- Male (8-10 week-old) wild-type and IL12p40-/- mice (n=15) on C57BL/6 background were infused with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the IL12p40-/- mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured by pulse wave velocity and atomic force microscopy. Histologically, IL12p40-/- mice exhibited increased maximal external diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA (small interfering RNA) augmented the Tgfß2-mediated Mmp2 expression in wild-type bone marrow-derived macrophages without affecting the expression of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or fibroblasts. Depletion of macrophages in IL12p40-/- mice by clodronate liposomes significantly decreased the maximal external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy. Conclusions- IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfß2.

Molecular characterization and functional analysis of IL-12p40 from Chinese sea bass (Lateolabrax maculatus) under biotic and abiotic stresses.

Interleukins are critical cytokines that are ubiquitously present in both vertebrates and invertebrates and constitute the front line of host innate immunity. Here, we identified and analyzed IL-12p40 from the Chinese sea bass Lateolabrax maculatus (LmIL-12p40). The LmIL-12p40 gene is expressed as a 1386-base pair transcript that encodes a polypeptide of 321 amino acids. Transcriptional expression analysis indicated that LmIL-12p40 mRNA was ubiquitously expressed in all tested tissues and had a comparatively high expression level in immune-associated tissues (head-kidney and intestines). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) experiments showed that, after Vibro harveyi and Streptococus agalactiae infection, LmIL-12p40 mRNA expression was significantly up-regulated in the spleen, liver and head-kidney. To further clarify the immune function of LmIL-12p40 after bacterial challenge, the recombinant LmIL-12p40 protein was acquired using a prokaryotic expression method. Furthermore, the LmIL-12p40 dimer (LmIL-12p80) could be produced via protein-protein interactions by incubating p40 monomer expressed from the pET28a vector (pET28a-LmIL-12p40) with p40 monomer expressed from the pGEX4T-1 vector (pGEX4T-1-LmIL-12p40). The antimicrobial activity of the purified LmIL-12p40 and LmIL-12p80 proteins were further studied in vitro using a bacterial growth inhibition test (for both liquid and solid cultures) and in vivo (using a bacterial growth inhibition test with the head-kidney tissues). Furthermore, BL21 (DE3) E. coli cells transformed with the recombinant pET28a-LmIL-12p40 vector were dramatically protected in response to metal toxicity and H2O2-related oxidative stress. In summary, this study will provide foundational information regarding the role of LmIL-12p40 in defending against various biotic and abiotic stresses in fishes, which should help to further clarify the functional mechanism of interleukins.

Clearance of apoptotic cells by macrophages induces regulatory phenotype and involves stimulation of CD36 and platelet-activating factor receptor.

Phagocytosis of apoptotic cells (efferocytosis) induces macrophage differentiation towards a regulatory phenotype (IL-10(high)/IL-12p40(low)). CD36 is involved in the recognition of apoptotic cells (AC), and we have shown that the platelet-activating factor receptor (PAFR) is also involved. Here, we investigated the contribution of PAFR and CD36 to efferocytosis and to the establishment of a regulatory macrophage phenotype. Mice bone marrow-derived macrophages were cocultured with apoptotic thymocytes, and the phagocytic index was determined. Blockage of PAFR with antagonists or CD36 with specific antibodies inhibited the phagocytosis of AC (~70-80%). Using immunoprecipitation and confocal microscopy, we showed that efferocytosis increased the CD36 and PAFR colocalisation in the macrophage plasma membrane; PAFR and CD36 coimmunoprecipitated with flotillin-1, a constitutive lipid raft protein, and disruption of these membrane microdomains by methyl-ß-cyclodextrin reduced AC phagocytosis. Efferocytosis induced a pattern of cytokine production, IL-10(high)/IL-12p40(low), that is, characteristic of a regulatory phenotype. LPS potentiated the efferocytosis-induced production of IL-10, and this was prevented by blocking PAFR or CD36. It can be concluded that phagocytosis of apoptotic cells engages CD36 and PAFR, possibly in lipid rafts, and this is required for optimal efferocytosis and the establishment of the macrophage regulatory phenotype.

Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models.

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.

Accelerated and progressive and lethal liver fibrosis in mice that lack interleukin (IL)-10, IL-12p40, and IL-13Rα2.

BACKGROUND & AIMS: Progressive fibrosis contributes to the morbidity of several chronic diseases; it typically develops slowly, so the mechanisms that control its progression and resolution have been difficult to model. The proteins interleukin (IL)-10, IL-12p40, and IL-13Rα2 regulate hepatic fibrosis following infection with the helminth parasite Schistosoma mansoni. We examined whether these mediators interact to slow the progression of hepatic fibrosis in mice with schistosomiasis. METHODS: IL-10(-/-), IL-12/23(p40)(-/-), and IL-13Rα2(-/-) mice were crossed to generate triple knockout (TKO) mice. We studied these mice to determine whether the simultaneous deletion of these 3 negative regulators of the immune response accelerated mortality from liver fibrosis following infection with S mansoni. RESULTS: Induction of inflammation by S mansoni, liver fibrosis, and mortality increased greatly in TKO mice compared with wild-type mice; 100% of the TKO mice died by 10 weeks after infection. Morbidity and mortality were associated with the development of portal hypertension, hepatosplenomegaly, gastrointestinal bleeding, ascites, thrombocytopenia, esophageal and gastric varices, anemia, and increased levels of liver enzymes, all features of advanced liver disease. IL-10, IL-12p40, and IL-13Rα2 reduced the production and activity of the profibrotic cytokine IL-13. A neutralizing antibody against IL-13 reduced the morbidity and mortality of the TKO mice following S mansoni infection. CONCLUSIONS: IL-10, IL-12p40, and IL-13Rα2 act cooperatively to suppress liver fibrosis in mice following infection with S mansoni. This model rapidly reproduces many of the complications observed in patients with advanced cirrhosis, so it might be used to evaluate the efficacy of antifibrotic reagents being developed for schistosomiasis or other fibrotic diseases associated with a T-helper 2 cell-mediated immune response.

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.

The emergence of TH17 cells as effectors of renal injury.

IL-17-producing Th17 effector cells directly induce renal inflammation by activating neutrophils or by participating in macrophage-mediated tissue injury. Th17 cells and cytokines participate in human and experimental renal disease, especially in proliferative glomerulonephritis where Th17 effector cells are active. Although growing evidence suggests Th17 cells are particularly relevant to effector responses involving neutrophils, there are still important questions to address before the complete functions of Th17 cells in renal disease are understood fully.

Proanthocyanidins inhibit UV-induced immunosuppression through IL-12-dependent stimulation of CD8+ effector T cells and inactivation of CD4+ T cells.

The inhibition of UVB-induced immunosuppression by dietary grape seed proanthocyanidins (GSP) has been associated with the induction of interleukin (IL)-12 in mice, and we now confirm that GSPs do not inhibit UVB-induced immunosuppression in IL-12p40 knockout (IL-12 KO) mice and that treatment of these mice with recombinant IL-12 restores the inhibitory effect. To characterize the cell population responsible for the GSP-mediated inhibition of UVB-induced immunosuppression and the role of IL-12 in this process, we used an adoptive transfer approach. Splenocytes and draining lymph nodes were harvested from mice that had been administered dietary GSPs (0.5%-1.0%, w/w), exposed to UVB, and sensitized by the application of 2,4-dinitrofluorobenzene (DNFB) onto the UVB-exposed skin. CD8(+) and CD4(+) T cells were positively selected and transferred into naive mice that were subsequently challenged by application of DNFB on the ear skin. Naive recipients that received CD8(+) T cells from GSP-treated, UVB-irradiated donors exhibited full contact hypersensitivity (CHS) response. Naive mice that received CD4(+) suppressor T cells from GSP-treated, UVB-exposed mice could mount a CHS response after sensitization and subsequent challenge with DNFB. On culture, the CD8(+) T cells from GSP-treated, UVB-exposed mice secreted higher levels (5- to 8-fold) of Th1 cytokines than CD8(+) T cells from UVB-irradiated mice not treated with GSPs. CD4(+) T cells from GSP-treated, UVB-exposed mice secreted significantly lower levels (80%-100%) of Th2 cytokines than CD4(+) T cells from UVB-exposed mice not treated with GSPs. These data suggest that GSPs inhibit UVB-induced immunosuppression by stimulating CD8(+) effector T cells and diminishing regulatory CD4(+) T cells.

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-12p40 and IL-18 play pivotal roles in orchestrating the cell-mediated immune response to a poxvirus infection.

A strong cell-mediated immune response is critical for controlling viral infections and is regulated by a number of cytokines, including IL-12 and IL-18. Indeed, some viruses have evolved to specifically target these pathways to counter the host immune response. Orthopoxviruses, including ectromelia virus, encode immune evasion molecules that specifically target IL-18 and IFN-gamma. We hypothesized that IL-12 and IL-18 are pivotal for induction of IFN-gamma production and subsequent generation of an effective host response to ectromelia virus infection. In this study, we demonstrate that absence of both IL-12p40 and IL-18 resulted in increased susceptibility to infection that was associated with skewing of the cytokine response to Th2 and a reduction in NK and CTL responses. The decrease in CTL response correlated with a defect in CD8(+) T cell proliferation and lower numbers of virus-specific CD8(+) T cells. Lack of either IL-12p40 and/or IL-18 was also associated with reduced numbers of CD8(+) T cells at sites of infection and with an increase in the numbers of splenic T regulatory cells. Taken together, our data indicate that IL-12p40 and IL-18 act in concert and play an important antiviral role through the up-regulation of IFN-gamma production and cell-mediated immune responses.

IL-12p40 is essential for the down-regulation of airway hyperresponsiveness in a mouse model of bronchial asthma with prolonged antigen exposure.

BACKGROUND: We previously reported a mouse model of bronchial asthma showing eosinophilic inflammation, but not airway hyperresponsiveness (AHR), after prolonged antigen exposure. This model showed an increase of IL-12 in the lung. OBJECTIVE: The aim of this study was to investigate the role of IL-12p40 in a murine asthma model with prolonged antigen exposures. METHODS: An ovalbumin (OVA)-induced asthma model was first established in wild-type (WT) and IL-12p40-deficient (IL-12p40(-/-)) mice. Both strains of mice were further exposed to either OVA (prolonged exposure group) or phosphate-buffered saline (positive control group) 3 days per week for 3 weeks. During week 4, both groups of mice were given a final challenge with OVA. RESULTS: Prolonged antigen exposures resulted in marked suppression of airway eosinophilia in both WT and IL-12p40(-/-) mice. However, AHR persisted in IL-12p40(-/-) but not in WT mice. There were no significant differences of IL-5, IL-13 or IFN-gamma levels in bronchoalveolar lavage fluid between WT and IL-12p40(-/-) mice. The hydroxyproline content of the lung and peribronchial fibrosis were, however, significantly increased in IL-12p40(-/-) mice. CONCLUSION: The results suggest that endogenous IL-12p40 is essential for inhibition of AHR and peribronchial fibrosis, but not eosinophilic inflammation, in a murine asthma model with prolonged antigen exposures.

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.

The molecular signature of oxidative metabolism and the mode of macrophage activation determine the shift from acute to chronic disease in experimental arthritis: critical role of interleukin-12p40.

OBJECTIVE: Repeated injection of streptococcal cell wall (SCW) fragments results in chronic arthritis in mice. The objective of this study was to identify genes and pathways that determine disease progression based on gene expression profiling in this model. METHODS: Chronic arthritis was induced in mice by 4 injections of SCW fragments. RNA samples were isolated from synovial tissue obtained at various time points and were analyzed using mouse genome array and quantitative reverse transcription-polymerase chain reaction techniques. The functional role of potential key genes was evaluated in mice with specific gene deletions. RESULTS: Gene expression analyses revealed a shift in molecular signature. In contrast to an up-regulation of the inflammatory response pathway, the pathways involved in oxidative metabolism were significantly down-regulated during the chronic phase of arthritis. Since oxidative metabolism determines the mode of macrophage activation, we investigated phenotype switching in macrophages. Markers of alternatively activated macrophages, such as arginase 1, were at maximal levels during acute inflammation. In contrast, induction of markers of classically activated macrophages (M1), such as interleukin-1beta (IL-1beta) and inducible nitric oxide synthase (iNOS), was relatively low during the acute phase of disease, but highly increased toward the chronic phase. M1 polarization during the chronic phase was accompanied by a Th1 signature, characterized by IL-12p40, IL-12p35, and interferon-gamma. However, the absence of IL-12p40, but not IL-12p35, significantly inhibited the chronic phase of arthritis and was marked by a reduction in IL-17 and iNOS levels, as well as restored expression of oxidative metabolism genes. CONCLUSION: M1 polarization accompanied by a decline in oxidative metabolism determine the chronic phase of arthritis. IL-12p40, most likely acting through the IL-23/IL-17 axis, plays a critical role in this process.

Interleukin-12p40 contributes to protection against lung injury after oral Yersinia enterocolitica infection.

OBJECTIVE: The impact of Yersinia enterocolitica on lung is incompletely understood, so we studied the inflammatory effects of Yersinia oral infection and the influence of IL-12p40 deficiency. METHODS: Wild-type (WT) and IL-12p40-/- (KO) mice were orally infected with Y. enterocolitica 0:3. After 3 and 21 days, cell viability in bronchoalveolar lavage (BAL) fluid, inflammatory reactions, lipid hydroperoxides, antioxidant enzyme expression and histological changes were studied. RESULTS: An effect on the lung was demonstrated by changes in lactate dehydrogenase, total protein (p <0.001), nitrosative stress and increase numbers of lymphocyte in the BAL fluid. All of these appeared to be IL-12 - independent since statistically significant changes in response to infection (at 21 days) did not differ between WT and KO groups. However, a protective role of IL-12 after infection was suggested by a decrease in cell viability, histopathological changes, different cell populations, higher lipid peroxidation and a decrease in antioxidant enzymes - glutathione peroxidase, superoxide dismutase-2 (p <0.05). The main changes were detected at day 21 suggesting a chronic effect of Yersinia infection and that IL-12 could play a role in the protection against chronic sequelae in the lung. CONCLUSIONS: These results demonstrate that Y. enterocolitica infection may induce inflammatory response in lung and that IL-12p40 could contribute to protection against lung injury.

Fat diet and alcohol-induced steatohepatitis after LPS challenge in mice: role of bioactive TNF and Th1 type cytokines.

Obesity with insulin resistance and alcohol are the most frequent causes of steatohepatitis. This work investigates the contribution of bioactive TNF and Th1 type cytokines in a mouse model of steatohepatitis induced by FAT alone or FAT+EtOH and endotoxin. The extent of liver injury and cytokine activation induced by endotoxin in chronic FAT-fed mice, FAT+EtOH-fed mice, or mice fed standard chow were analyzed. Endotoxin administration to either FAT-fed or FAT+EtOH-fed mice increased serum ALT and AST compared to standard chow mice. Immunoreactive TNF was strongly activated by LPS in FAT-fed and FAT+EtOH-fed mice which presented the highest levels, but low levels were found in standard chow mice. In contrast, bioactive TNF was only present in serum of FAT-fed and in particular the highest levels were found in FAT+EtOH-fed mice. Moreover, soluble TNFR2 but not TNFR1 was found in lower amounts in serum of FAT+EtOH-fed mice compared to FAT-fed mice. Steatohepatitis was associated with increased IL-6, IFN-gamma, and iNOS mRNA and proteins. Data show that a moderately FAT diet and low-dose EtOH concur to generate steatohepatitis and TNF liver expression after LPS. In this model, changes in the regulation of TNF are associated with increased expression of IL-6, IFN-gamma, and iNOS.

Role of cytokine p40 family in multiple sclerosis.

Over the last couple of decades of neuro-immunological research, the p40 family of cytokines has emerged out as one of the most intriguing areas of interest because of multi-faceted roles of these cytokine in immune-modulation and inflammation. The IL-12, the most widely studied cytokine of this family, is well-characterized for its Th1-favoring activity, and therefore plays a key role in the pathophysiology of Th1-mediated autoimmune diseases like multiple sclerosis (MS). On the other hand, the IL-23, another member of the p40 family with shared p40 subunit, drives polarization of Th17, a subset of T cell suspected to have a key role in the pathophysiology of MS and experimental allergic encephalomyelitis (EAE), poses a challenge to our current understanding of Th1/Th2 hypotheses in autoimmune diseases. However, the more puzzling issues, the researchers are currently confronted with, are the biological roles of other two members of this family, the p40 monomer and the p40(2), the homodimer. Predominance of the mRNA level of p40 over p35 in the central nervous system of EAE and MS suggests a possible involvement of p40 in the pathogenesis of MS. However, the distinctive biological role of monomeric and dimeric form of p40 is not clearly understood yet. Initially, it was thought that p402 does not have any biological activity and only involved in antagonizing bioactive IL-12 but according to recent evidences, both p402 and p40 appear to have a proinflammatory role, therefore might be a crucial molecule in the pathogenesis of MS. The current review focuses on biological function of p40 family of cytokines with particular emphasis on MS.

Deviation from a strong Th1-dominated to a modest Th17-dominated CD4 T cell response in the absence of IL-12p40 and type I IFNs sustains protective CD8 T cells.

The differentiation of naive CD4 T cells into specific effector subsets is controlled in large part by the milieu of cytokines present during their initial encounter with Ag. Cytokines that drive differentiation of the newly described Th17 lineage have been characterized in vitro, but the cytokines that prime commitment to this lineage in response to infection in vivo are less clear. Listeria monocytogenes (Lm) induces a strong Th1 response in wild-type mice. By contrast, we demonstrate that in the absence of IL-12p40 (or IFN-gamma) and type I IFN receptor signaling, the Th1 Ag-specific CD4 T cell response is virtually abolished and replaced by a relatively low magnitude Th17-dominated response. This Th17 response was dependent on TGF-beta and IL-6. Despite this change in CD4 T cell response, neither the kinetics of the CD4 and CD8 T cell responses, the quality of the CD8 T cell response, nor the ability of CD8 T cells to mediate protection were affected. Thus, generation of protective CD8 T cell immunity was resilient to perturbations that replace a strong Th1-dominated to a reduced magnitude Th17-dominated Ag-specific CD4 T cell response.

IL-23 is required for neutrophil homeostasis in normal and neutrophilic mice.

IL-23 is secreted by macrophages and dendritic cells in response to microbial products and inflammatory cytokines. IL-23 is a heterodimer composed of the unique IL-23p19 subunit linked to the common p40 subunit that it shares with IL-12. IL-23 is implicated in autoimmune diseases, where it supports the expansion of IL-17A-producing CD4+ Th17 cells. IL-23 also regulates granulopoiesis in a neutrostat regulatory feedback loop through IL-17A-producing neutrophil regulatory (Tn) cells, most of which express gammadelta TCR. This homeostatic system is disrupted in mice lacking adhesion molecules like beta2-integrins (Itgb2-/-) which have defective neutrophil trafficking and neutrophilia. To test the role of IL-23 in the homeostatic regulation of circulating neutrophil numbers, we measured blood neutrophil numbers in p40-deficient (IL12b-/-) mice and found them reduced compared with wild-type mice. IL12b-/-Itgb2-/- mice, lacking beta2-integrins, IL-12, and IL-23 showed significantly blunted neutrophilia compared with Itgb2-/- mice. Treatment of both IL12b-/- and IL12b-/-Itgb2-/- mice with IL-23, but not IL-12, restored circulating neutrophil counts. Serum levels of IL-17A were readily detectable in Itgb2-/- mice, but not in IL12b-/-Itgb2-/- mice, suggesting that IL-17A production is reduced when IL-23 is absent. Similarly, tissue mRNA expression of IL-17A was reduced in IL12b-/-Itgb2-/-mice compared with Itgb2-/- controls. The total number of CD3+ IL-17A-producing Tn cells were significantly reduced in the spleen and lamina propria of IL12b-/-Itgb2-/- mice, with the largest reduction found in gammadelta+ T cells. Our results suggest a prominent role of IL-23 in the regulation of granulopoiesis and the prevalence of IL-17A-producing Tn cells.