Interleukin 4 promotes anti-inflammatory macrophages that clear cartilage debris and inhibits osteoclast development to protect against osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the leading cause of joint failure, is characterized by breakdown of articular cartilage and remodeling of subchondral bone in synovial joints. Despite the high prevalence and debilitating effects of OA, no disease-modifying drugs exist. Increasing evidence, including genetic variants of the interleukin 4 (IL-4) and IL-4 receptor genes, implicates a role for IL-4 in OA, however, the mechanism underlying IL-4 function in OA remains unknown. Here, we investigated the role of IL-4 in OA pathogenesis. METHODS: Il4-, myeloid-specific-Il4ra-, and Stat6-deficient and control mice were subjected to destabilization of the medial meniscus to induce OA. Macrophages, osteoclasts, and synovial explants were stimulated with IL-4 in vitro, and their function and expression profiles characterized. RESULTS: Mice lacking IL-4, IL-4Ra in myeloid cells, or STAT6 developed exacerbated cartilage damage and osteophyte formation relative to WT controls. In vitro analyses revealed that IL-4 downregulates osteoarthritis-associated genes, enhances macrophage phagocytosis of cartilage debris, and inhibits osteoclast differentiation and activation via the type I receptor. CONCLUSION: Our findings demonstrate that IL-4 protects against osteoarthritis in a myeloid and STAT6-dependent manner. Further, IL-4 can promote an immunomodulatory microenvironment in which joint-resident macrophages polarize towards an M2 phenotype and efficiently clear pro-inflammatory debris, and osteoclasts maintain a homeostatic level of activity in subchondral bone. These findings support a role for IL-4 modulation of myeloid cell types in maintenance of joint health and identify a pathway that could provide therapeutic benefit for osteoarthritis.

M2-like, dermal macrophages are maintained via IL-4/CCL24-mediated cooperative interaction with eosinophils in cutaneous leishmaniasis.

Tissue-resident macrophages (TRMs) maintain tissue homeostasis, but they can also provide a replicative niche for intracellular pathogens such as Leishmania How dermal TRMs proliferate and maintain their M2 properties even in the strong TH1 environment of the L. major infected dermis is not clear. Here, we show that, in infected mice lacking IL-4/13 from eosinophils, dermal TRMs shifted to a proinflammatory state, their numbers declined, and disease was attenuated. Intravital microscopy revealed a rapid infiltration of eosinophils followed by their tight interaction with dermal TRMs. IL-4-stimulated dermal TRMs, in concert with IL-10, produced a large amount of CCL24, which functioned to amplify eosinophil influx and their interaction with dermal TRMs. An intraperitoneal helminth infection model also demonstrated a requirement for eosinophil-derived IL-4 to maintain tissue macrophages through a CCL24-mediated amplification loop. CCL24 secretion was confined to resident macrophages in other tissues, implicating eosinophil-TRM cooperative interactions in diverse inflammatory settings.

IL-4/IL-13-mediated polarization of renal macrophages/dendritic cells to an M2a phenotype is essential for recovery from acute kidney injury.

Cytokines IL-4 and IL-13 play important roles in polarization of macrophages/dendritic cells to an M2 phenotype, which is important for recovery from acute kidney injury. Both IL-4 and IL-13 activate JAK3/STAT6 signaling. In mice with diphtheria toxin receptor expression in proximal tubules (selective injury model), a relatively selective JAK3 inhibitor, tofacitinib, led to more severe kidney injury, delayed recovery from acute kidney injury, increased inflammatory M1 phenotype markers and decreased reparative M2 phenotype markers of macrophages/dendritic cells, and development of more severe renal fibrosis after diphtheria toxin administration. Similarly, there was delayed recovery and increased tubulointerstitial fibrosis in these diphtheria toxin-treated mice following tamoxifen-induced deletion of both IL-4 and IL-13, with increased levels of M1 and decreased levels of M2 markers in the macrophages/dendritic cells. Furthermore, deletion of IL-4 and IL-13 led to a decrease of tissue reparative M2a phenotype markers but had no effect on anti-inflammatory M2c phenotype markers. Deletion of IL-4 and IL-13 also inhibited recovery from ischemia-reperfusion injury in association with increased M1 and decreased M2 markers and promoted subsequent tubulointerstitial fibrosis. Thus, IL-4 and IL-13 are required to effectively polarize macrophages/dendritic cells to an M2a phenotype and to promote recovery from acute kidney injury.

Conditional IL-4/IL-13-deficient mice reveal a critical role of innate immune cells for protective immunity against gastrointestinal helminths.

Approximately one-third of the world population is infected with gastrointestinal helminths. Studies in mouse models have demonstrated that the cytokines interleukin (IL)-4 and IL-13 are essential for worm expulsion, but the critical cellular source of these cytokines is poorly defined. Here, we compared the immune response to Nippostrongylus brasiliensis in wild-type, T cell-specific IL-4/IL-13-deficient and general IL-4/IL-13-deficient mice. We show that T cell-derived IL-4/IL-13 promoted T helper 2 (Th2) polarization in a paracrine manner, differentiation of alternatively activated macrophages, and tissue recruitment of innate effector cells. However, innate IL-4/IL-13 played the critical role for induction of goblet cell hyperplasia and secretion of effector molecules like Mucin5ac and RELMß in the small intestine. Surprisingly, T cell-specific IL-4/IL-13-deficient and wild-type mice cleared the parasite with comparable efficiency, whereas IL-4/IL-13-deficient mice showed impaired expulsion. These findings demonstrate that IL-4/IL-13 produced by cells of the innate immune system is required and sufficient to initiate effective type 2 immune responses resulting in protective immunity against N. brasiliensis.

Interleukin 4 Deficiency Reverses Development of Secondary Pseudomonas aeruginosa Pneumonia During Sepsis-Associated Immunosuppression.

BACKGROUND: Interleukin 4 (IL-4) is an important cytokine that may modulate development of secondary bacterial pneumonia during sepsis-induced immunosuppression. METHODS: We established an experimental model of cecal ligation and puncture (CLP)-induced sublethal polymicrobial sepsis followed by secondary Pseudomonas aeruginosa pulmonary infection, RESULTS: IL-4-deficient mice that underwent CLP were resistant to secondary pulmonary P. aeruginosa infection. As compared to wild-type mice, IL-4 knockout (KO) mice displayed improved survival and better bacterial clearance. Furthermore, IL-4 KO mice exhibited enhanced lung inflammation, neutrophil recruitment to airspaces, and elevated pulmonary cytokine production, with significantly increased tumor necrosis factor α (TNF-α) production. Neutralization of TNF-α could reverse the enhanced protection against secondary P. aeruginosa pneumonia in septic IL-4 KO mice, indicating that the resistance of septic IL-4 KO mice to secondary bacterial pneumonia was partially mediated by TNF-α. In addition, IL-4 priming displayed marked impairment of the ability of alveolar macrophages to phagocytose and kill P. aeruginosa in vitro, and this defect was associated with decreased activation of Akt, JNK, p38MAPK, and ERK intracellular signaling pathways by IL-4. Finally, neutralization of IL-4 in septic mice could improve survival and clearance of bacteria from the lungs of septic mice infected with P. aeruginosa. CONCLUSIONS: Our findings provide new insight for immunopathologic mechanisms of sepsis-induced secondary bacterial pneumonia.

Basophil-derived interleukin-4 controls the function of natural helper cells, a member of ILC2s, in lung inflammation.

Allergic asthma is an inflammatory disease characterized by lung eosinophilia controlled by type 2 cytokines. Cysteine proteases are potent triggers of allergic inflammation by causing barrier disruption in lung epithelial cells inducing the elevation of interleukin-5 (IL-5) and IL-13 from natural helper (NH) cells, a member of ILC2s, which leads to lung eosinophilia. In this study, we found that basophils play a crucial role in NH cell-mediated eosinophilic inflammation induced by protease allergens. Conditional deletion of basophils caused a resolution of the papain-induced eosinophilia and mucus production. Resolution of eosinophilia was also observed in mice lacking IL-4 specifically in basophils, indicating that basophil-derived IL-4 enhanced expression of the chemokine CCL11, as well as IL-5, IL-9, and IL-13 in NH cells, thus attracting eosinophils. These results demonstrate that IL-4 from basophils has an important role in the NH-derived cytokine and chemokine expression, subsequently leading to protease allergen-induced airway inflammation.

Activation of invariant natural killer T cells impedes liver regeneration by way of both IFN-γ- and IL-4-dependent mechanisms.

UNLABELLED: Invariant natural killer T (iNKT) cells are a major subset of lymphocytes found in the liver. These cells mediate various functions, including hepatic injury, fibrogenesis, and carcinogenesis. However, the function of iNKT cells in liver regeneration remains unclear. In the present study, partial hepatectomy (PHx) was used to study liver regeneration. α-Galactosylceramide (α-GalCer), a specific ligand for iNKT cells, was used to induce iNKT cell activation. After PHx, two strains of iNKT cell-deficient mice, CD1d(-/-) and Jα281(-/-) mice, showed normal liver regeneration. Injection of α-GalCer before or after PHx, which rapidly stimulated interferon-gamma (IFN-γ) and interleukin (IL)-4 production by iNKT cells, markedly inhibited liver regeneration. In vitro treatment with IFN-γ inhibited hepatocyte proliferation. In agreement with this in vitro finding, genetic disruption of IFN-γ or its downstream signaling molecule signal transducer and activator of transcription (STAT)1 significantly abolished the α-GalCer-mediated inhibition of liver regeneration. In vitro exposure to IL-4 did not affect hepatocyte proliferation, but surprisingly, genetic ablation of IL-4 or its downstream signaling molecule STAT6 partially eliminated the inhibitory effect of α-GalCer on liver regeneration. Further studies revealed that IL-4 contributed to α-GalCer-induced iNKT cell expansion and IFN-γ production, thereby inhibiting liver regeneration. CONCLUSION: iNKT cells play a minor role in controlling liver regeneration after PHx under healthy conditions. Activation of iNKT cells by α-GalCer induces the production of IFN-γ, which directly inhibits liver regeneration, and IL-4, which indirectly attenuates liver regeneration by stimulating iNKT cell expansion and IFN-γ production.

IL-4 and IL-4 receptor expression is dispensable for the development and function of natural killer T cells.

CD4 T cells acquire functional properties including cytokine production upon antigenic stimulation through the T cell receptor (TCR) and differentiate into T helper (Th) cells. Th1 cells produce interferon (IFN)-γ and Th2 cells produce interleukin (IL)-4. Th1 and 2 cells utilize IFN-γ and IL-4 for further maturation and maintenance, respectively. Promyelocytic leukemia zinc finger (PLZF)-expressing invariant natural killer T (iNKT) cells develop in the thymus and acquire functional ability to produce IL-4 and IFN-γ in the thymus in the absence of antigenic stimulation. In response to antigenic stimulation, iNKT cells rapidly produce IFN-γ and IL-4. However, it is still unknown as to whether iNKT cells require these cytokines for maturation or survival in vivo. In this study, using IL-4- and IL-4 receptor- (IL-4R) deficient mice, we demonstrate that IL-4 as well as IL-4R expression is dispensable for the development, function and maintenance of iNKT cells.

Shp1 regulates T cell homeostasis by limiting IL-4 signals.

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell­intrinsic role of Shp1, we characterized mice with a T cell­specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1(fl/fl) CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1- deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.

Invariant NKT cell activation induces neutrophil accumulation and hepatitis: opposite regulation by IL-4 and IFN-γ.

UNLABELLED: Alpha-Galactosylceramide (α-Galcer), a specific agonist for invariant natural killer T (iNKT) cells, is being evaluated in clinical trials for the treatment of viral hepatitis and liver cancer. However, the results from α-Galcer treatment are mixed, partially because of the variety of cytokines produced by activated iNKT cells that have an unknown synergistic effect on the progression of liver disease. It is well documented that injection of α-Galcer induces mild hepatitis with a rapid elevation in the levels of interleukin (IL)-4 and a delayed elevation in the levels of interferon-gamma (IFN-γ), and both of these cytokines are thought to mediate many functions of iNKT cells. Surprisingly, genetic deletion of both IL-4 and IFN-γ aggravated, rather than abolished, α-Galcer-induced iNKT hepatitis. Moreover, genetic ablation of IL-4, the IL-4 receptor, or its downstream signaling molecule signal transducer and activator of transcription (STAT)6 ameliorated α-Galcer-induced neutrophil infiltration, liver injury, and hepatitis. In contrast, genetic deletion of IFN-γ, the IFN-γ receptor, or its downstream signaling molecule STAT1 enhanced liver neutrophil accumulation, thereby exacerbating liver injury and hepatitis. Moreover, depletion of neutrophils eradicated α-Galcer-induced liver injury in wild-type, STAT1 knockout, and IFN-γ knockout mice. CONCLUSION: Our results propose a model in which activated iNKT cells rapidly release IL-4, which promotes neutrophil survival and hepatitis but also sequentially produce IFN-γ, which acts in a negative feedback loop to ameliorate iNKT hepatitis by inducing neutrophil apoptosis. Thus, modification of iNKT production of IL-4 and IFN-γ may have the potential to improve the efficacy of α-Galcer in the treatment of liver disease.

Thymic stromal lymphopoietin amplifies the differentiation of alternatively activated macrophages.

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) has been associated with the promotion of type 2 inflammation and the induction of allergic disease. In humans TSLP is elevated in the lungs of asthma patients and in the lesional skin of individuals with atopic dermatitis, whereas mice lacking TSLP responses are refractory to models of Th2-driven allergic disease. Although several cell types, including dendritic cells, basophils, and CD4 T cells, have been shown to respond to TSLP, its role in macrophage differentiation has not been studied. Type 2 cytokines (i.e., IL-4 and IL-13) can drive the differentiation of macrophages into alternatively activated macrophages (aaMs, also referred to as M2 macrophages). This population of macrophages is associated with allergic inflammation. We therefore reasoned that TSLP/TSLPR signaling may be involved in the differentiation and activation of aaMs during allergic airway inflammation. In this study, we report that TSLP changes the quiescent phenotype of pulmonary macrophages toward an aaM phenotype during TSLP-induced airway inflammation. This differentiation of airway macrophages was IL-13-, but not IL-4-, dependent. Taken together, we demonstrate in this study that TSLP/TSLPR plays a significant role in the amplification of aaMΦ polarization and chemokine production, thereby contributing to allergic inflammation.

MyD88 signaling pathway is involved in renal fibrosis by favoring a TH2 immune response and activating alternative M2 macrophages.

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4(+) T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (T(H)2)-prone inflammatory milieu in a MyD88-dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished T(H)2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10(+) and CD206(+) CD11b(high) cells, at 7 d after surgery. We evaluated the role of a T(H)2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF-ß levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and T(H)1:T(H)2 balance, as T(H)2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.

Endogenous interleukin-4 regulates glutathione synthesis following acetaminophen-induced liver injury in mice.

In a recent study, we reported that interleukin (IL)-4 had a protective role against acetaminophen (APAP)-induced liver injury (AILI), although the mechanism of protection was unclear. Here, we carried out more detailed investigations and have shown that one way IL-4 may control the severity of AILI is by regulating glutathione (GSH) synthesis. In the present studies, the protective role of IL-4 in AILI was established definitively by showing that C57BL/6J mice made deficient in IL-4 genetically (IL-4(-/-)) or by depletion with an antibody, were more susceptible to AILI than mice not depleted of IL-4. The increased susceptibility of IL-4(-/-) mice was not due to elevated levels of hepatic APAP-protein adducts but was associated with a prolonged reduction in hepatic GSH that was attributed to decreased gene expression of γ-glutamylcysteine ligase (γ-GCL). Moreover, administration of recombinant IL-4 to IL-4(-/-) mice postacetaminophen treatment diminished the severity of liver injury and increased γ-GCL and GSH levels. We also report that the prolonged reduction of GSH in APAP-treated IL-4(-/-) mice appeared to contribute toward increased liver injury by causing a sustained activation of c-Jun-N-terminal kinase (JNK) since levels of phosphorylated JNK remained significantly higher in the IL-4(-/-) mice up to 24 h after APAP treatment. Overall, these results show for the first time that IL-4 has a role in regulating the synthesis of GSH in the liver under conditions of cellular stress. This mechanism appears to be responsible at least in part for the protective role of IL-4 against AILI in mice and may have a similar role not only in AILI in humans but also in pathologies of the liver caused by other drugs and etiologies.

The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice.

CD4(+) T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 µm vs. 294.4 ± 72.2 µm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.

Interleukin-4 (IL-4) and IL-13 suppress excessive neutrophil infiltration and hepatocyte damage during acute murine schistosomiasis japonica.

Due to the importance of neutrophils and proinflammatory cytokines in schistosomal liver damage, we analyzed the mechanisms underlying neutrophil and proinflammatory responses in murine schistosomiasis japonica. We found that granulomatous inflammation around parasite eggs in the liver was greater in Schistosoma japonicum-infected IL-4-/- IL-13-/- (double-knockout [DKO]) mice than in infected wild-type (WT) mice at 6 weeks, but not at 8 weeks, postinfection, suggesting the importance of Th2 responses in these typical hepatic lesions. Infected DKO mice also showed increased neutrophil infiltration accompanying more severe pathology, as shown by the enhanced necrosis of hepatocytes. This was not likely due to a Th1/Th2 imbalance, because there was no detectable increase in gamma interferon (IFN-γ) production in these DKO mice. mRNA expression of interleukin-17A (IL-17A), proinflammatory cytokines, and the neutrophil chemoattractant CXCL2 in liver was higher in infected DKO mice than in WT mice. However, in IL-4-/- IL-13-/- IL-17A-/- (triple-knockout [TKO]) mice, the absence of IL-17A was associated with only marginal differences in schistosomal liver damage, suggesting that IL-17A is only partially responsible for neutrophil-driven hepatic damage. Furthermore, the expression of mRNAs encoding proinflammatory cytokines was not under the control of IL-17A in TKO mice. These findings indicate that IL-4 and IL-13 suppress excessive neutrophil recruitment, proinflammatory cytokine production, and hepatic damage during the acute stage of S. japonicum infection, suggesting that neutrophils and proinflammatory cytokines are mainly responsible for hepatocyte damage during acute murine schistosomiasis japonica. However, neutrophil induction and the production of proinflammatory cytokines were not due solely to IL-17A.

IL-4 deficiency is associated with mechanical hypersensitivity in mice.

Interleukin-4 (IL-4) is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko) mice to characterize their pain behavior before and after chronic constriction injury (CCI) of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS) of IL-4 ko mice in comparison with wildtype (wt) mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001), while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF), IL-1ß, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1ß gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001) 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014). Remarkably, CCI induced TNF (p<0.01), IL-1ß (p<0.05), IL-10 (p<0.05), and IL-13 (p<0.001) gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of µ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion.

Regulatory role of Vγ1 γδ T cells in tumor immunity through IL-4 production.

It has been demonstrated that the two main subsets of peripheral γδ T cells, Vγ1 and Vγ4, have divergent functions in many diseases models. Recently, we reported that Vγ4 γδ T cells played a protective role in tumor immunity through eomesodermin-controlled mechanisms. However, the precise roles of Vγ1 γδ T cells in tumor immunity, especially whether Vγ1 γδ T cells have any interaction with Vγ4 γδ T cells, remain unknown. We demonstrated in this paper that Vγ1 γδ T cells suppressed Vγ4 γδ T cell-mediated antitumor function both in vitro and in vivo, and this suppression was cell contact independent. Using neutralizing anti-IL-4 Ab or IL-4(-/-) mice, we determined the suppressive factor derived from Vγ1 γδ T cells was IL-4. Indeed, treatment of Vγ4 γδ T cells with rIL-4 significantly reduced expression levels of NKG2D, perforin, and IFN-γ. Finally, Vγ1 γδ T cells produced more IL-4 and expressed significantly higher level of GATA-3 upon Th2 priming in comparison with Vγ4 γδ T cells. Therefore, to our knowledge, our results established for the first time a negative regulatory role of Vγ1 γδ T cells in Vγ4 γδ T cell-mediated antitumor immunity through cell contact-independent and IL-4-mediated mechanisms. Selective depletion of this suppressive subset of γδ T cells may be beneficial for tumor immune therapy.

Cell-free antigens from Paracoccidioides brasiliensis drive IL-4 production and increase the severity of paracoccidioidomycosis.

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

Effect of cytokine interplay on macrophage polarization during chronic pulmonary infection with Cryptococcus neoformans.

The immune response to Cryptococcus neoformans following pulmonary infection of C57BL/6 wild-type (WT) mice results in the development of persistent infection with characteristics of allergic bronchopulmonary mycosis (ABPM). To further clarify the role of Th1/Th2 polarizing cytokines in this model, we performed kinetic analysis of cytokine responses and compared cytokine profiles, pathologies, and macrophage (Mac) polarization status in C. neoformans-infected WT, interleukin-4-deficient (IL-4(-/-)), and gamma interferon-deficient (IFN-γ(-/-)) C57BL/6 mice. Results show that cytokine expression in the infected WT mice is not permanently Th2 biased but changes dynamically over time. Using multiple Mac activation markers, we further demonstrate that IL-4 and IFN-γ regulate the polarization state of Macs in this model. A higher IL-4/IFN-γ ratio leads to the development of alternatively activated Macs (aaMacs), whereas a higher IFN-γ/IL-4 ratio leads to the generation of classically activated Macs (caMacs). WT mice that coexpress IL-4 and IFN-γ during fungal infection concurrently display both types of Mac polarization markers. Concurrent stimulation of Macs with IFN-γ and IL-4 results in an upregulation of both sets of markers within the same cells, i.e., formation of an intermediate aaMac/caMac phenotype. These cells express both inducible nitric oxide synthase (important for clearance) and arginase (associated with chronic/progressive infection). Together, our data demonstrate that the interplay between Th1 and Th2 cytokines supports chronic infection, chronic inflammation, and the development of ABPM pathology in C. neoformans-infected lungs. This cytokine interplay modulates Mac differentiation, including generation of an intermediate caMac/aaMac phenotype, which in turn may support chronic "steady-state" fungal infection and the resultant ABPM pathology.

Biochemical and genetic evidence for a SAP-PKC-theta interaction contributing to IL-4 regulation.

Signaling lymphocytic activation molecule-associated protein (SAP), an adaptor molecule that recruits Fyn to the signaling lymphocytic activation molecule (SLAM) family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4(+) T cells from SAP-deficient mice have defective TCR-induced and follicular Th cell IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4(+) T cells exhibit decreased protein kinase C (PKC)-theta recruitment upon TCR stimulation. We demonstrate in this paper using GST pulldowns and coimmunoprecipitation studies that SAP constitutively associates with PKC- in T cells. SAP-PKC-theta interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP's interactions with PKC-theta occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-theta recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-theta, like SAP, was required for SLAM-mediated increases in IL-4 production, and, conversely, membrane-targeted PKC-theta mutants rescued IL-4 expression in SAP(-/-) CD4(+) T cells, providing genetic evidence that PKC-theta is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production.