Reduced TCR‐dependent activation through citrullination of a T‐cell epitope enhances Th17 development by disruption of the STAT3/5 balance

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4⁺ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.     

Inhibition of Interferon Regulatory Factor 4 Suppresses Th1 and Th17 Cell Differentiation and Ameliorates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T‐cell‐mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. Recent study demonstrated that IRF4 was critical for the generation of IL‐17‐producing Th17 cells. However, the effect of IRF4 on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, needs to be further investigated. In our current study, inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC‐T‐cell coculture system, SiIRF4‐treated DCs resulted in significantly less IFN‐γ and IL‐17 production from T cells. Next, we adoptively transfer CD11c⁺ DCs from SiIRF4‐treated mice into recipient mice and found that these CD11c⁺ DCs ameliorated EAE. Furthermore, CD11c⁺ DCs from SiIRF4‐treated naive mice exhibited significantly reduced expression of pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and IL‐12/IL‐23 (p40), and a corresponding increase in anti‐inflammatory IL‐10 expression. In conclusion, inhibition of IRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE, via a direct regulation of DCs.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

Boswellic acids reduce Th17 differentiation via blockade of IL‐1β‐mediated IRAK1 signaling

Interferon‐gamma producing CD4⁺ T (Th1) cells and IL‐17‐producing CD4⁺ T (Th17) cells are involved in the pathogenesis of several autoimmune diseases including multiple sclerosis. Therefore, the development of treatment strategies controlling the generation and expansion of these effector cells is of high interest. Frankincense, the resin from trees of the genus Boswellia, and particularly its prominent bioactive compound acetyl‐11‐keto‐β‐boswellic acid (AKBA), have potent anti‐inflammatory properties. Here, we demonstrate that AKBA is able to reduce the differentiation of human CD4⁺ T cells to Th17 cells, while slightly increasing Th2‐ and Treg‐cell differentiation. Furthermore, AKBA reduces the IL‐1β‐triggered IL‐17A release of memory Th17 cells. AKBA may affect IL‐1β signaling by preventing IL‐1 receptor‐associated kinase 1 phosphorylation and subsequently decreasing STAT3 phosphorylation at Ser727, which is required for Th17‐cell differentiation. The effects of AKBA on Th17 differentiation and IL‐17A release make the compound a good candidate for potential treatment of Th17‐driven diseases.     

Lck regulates IL-10 expression in memory-like Th1 cells

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck^−/− Th1 skewed cells have normal expression of T‐bet and produce IFN‐γ at WT levels. However, there is a 3‐fold increase in IL‐10 producing cells in the mutant cultures. These cells do not have elevated levels of IL‐4, GATA3, IL‐17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3⁺ T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL‐10 in the lck^−/− Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L⁺ (naïve) cells are similar to WT cells. Furthermore, this IL‐10 expression appears to be dependent on IL‐12 and correlates with elevated c‐Maf. These data highlight a previously unappreciated role for Lck in regulating IL‐10 in Th1 cells.     

T‐bet protects against exacerbation of schistosome egg‐induced immunopathology by regulating Th17‐mediated inflammation

C57BL/6 mice infected with Schistosoma mansoni naturally develop mild CD4⁺ T‐cell‐mediated immunopathology characterized by small hepatic granulomas around parasite eggs. However, immunization with soluble egg Ag in CFA markedly exacerbates the lesions by inducing a potent proinflammatory environment with high levels of IFN‐γ and IL‐17, which are signature cytokines of distinct Th1‐ versus Th17‐cell lineages. To determine the relative role of these subsets in disease exacerbation, we examined mice deficient in T‐bet (T‐bet^?/?), which is required for Th1 differentiation and IFN‐γ production. We now report that immunization with soluble egg Ag in CFA caused a significantly greater enhancement of egg‐induced hepatic immunopathology in T‐bet^?/? mice compared with WT controls, and analysis of their granulomas disclosed a higher proportion of activated DC and CD4⁺ T cells, as well as a marked influx of neutrophils. The absence of IFN‐γ in the T‐bet^?/? mice correlated with a marked increase in IL‐23p19, IL‐17 and TNF‐α in granulomas and MLN. In contrast, T‐bet^?/? mice had lower levels of IL‐4, IL‐5 and IL‐10 and a reduction in FIZZ1 and FoxP3 expression, suggesting diminished regulatory activity, respectively, by alternatively activated macrophages and Treg. These findings demonstrate that T‐bet‐dependent signaling negatively regulates Th17‐mediated immunopathology in severe schistosomiasis.     

IL‐6 controls susceptibility to helminth infection by impeding Th2 responsiveness and altering the Treg phenotype in vivo

IL‐6 plays a pivotal role in favoring T‐cell commitment toward a Th17 cell rather than Treg‐cell phenotype, as established through in vitro model systems. We predicted that in the absence of IL‐6, mice infected with the gastrointestinal helminth Heligmosomoides polygyrus would show reduced Th17‐cell responses, but also enhanced Treg‐cell activity and consequently greater susceptibility. Surprisingly, worm expulsion was markedly potentiated in IL‐6‐deficient mice, with significantly stronger adaptive Th2 responses in both IL‐6^?/? mice and BALB/c recipients of neutralizing anti‐IL‐6 monoclonal Ab. Although IL‐6‐deficient mice showed lower steady‐state Th17‐cell levels, IL‐6‐independent Th17‐cell responses occurred during in vivo infection. We excluded the Th17 response as a factor in protection, as Ab neutralization did not modify immunity to H. polygyrus infection in BALB/c mice. Resistance did correlate with significant changes to the associated Treg‐cell phenotype however, as IL‐6‐deficient mice displayed reduced expression of Foxp3, Helios, and GATA‐3, and enhanced production of cytokines within the Treg‐cell population. Administration of an anti‐IL‐2:IL‐2 complex boosted Treg‐cell proportions in vivo, reduced adaptive Th2 responses to WT levels, and fully restored susceptibility to H. polygyrus in IL‐6‐deficient mice. Thus, in vivo, IL‐6 limits the Th2 response, modifies the Treg‐cell phenotype, and promotes host susceptibility following helminth infection.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Pathogenic T helper type 17 cells contribute to type 1 diabetes independently of interleukin‐22

We have shown that pathogenic T helper type 17 (Th17) cells differentiated from naive CD4⁺ T cells of BDC2·5 T cell receptor transgenic non‐obese diabetic (NOD) mice by interleukin (IL)‐23 plus IL‐6 produce IL‐17, IL‐22 and induce type 1 diabetes (T1D). Neutralizing interferon (IFN)‐γ during the polarization process leads to a significant increase in IL‐22 production by these Th17 cells. We also isolated IL‐22‐producing Th17 cells from the pancreas of wild‐type diabetic NOD mice. IL‐27 also blocked IL‐22 production from diabetogenic Th17 cells. To determine the functional role of IL‐22 produced by pathogenic Th17 cells in T1D we neutralized IL‐22 in vivo by using anti‐IL‐22 monoclonal antibody. We found that blocking IL‐22 did not alter significantly adoptive transfer of disease by pathogenic Th17 cells. Therefore, IL‐22 is not required for T1D pathogenesis. The IL‐22Rα receptor for IL‐22 however, increased in the pancreas of NOD mice during disease progression and based upon our and other studies we suggest that IL‐22 may have a regenerative and protective role in the pancreatic islets.     

TGF‐β indirectly favors the development of human Th17 cells by inhibiting Th1 cells

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti‐proliferative effect of TGF‐β than Th1 and Th2 clones or circulating Th1‐oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl‐2 expression and reduced apoptosis in the presence of TGF‐β, in comparison with Th1 cells. Umbilical cord blood naïve CD161⁺CD4⁺ T cells, which contain the precursors of human Th17 cells, differentiated into IL‐17A‐producing cells only in response to IL‐1β plus IL‐23, even in serum‐free cultures. TGF‐β had no effect on constitutive RORγt expression by umbilical cord blood CD161⁺ T cells but it increased the relative proportions of CD161⁺ T cells differentiating into Th17 cells in response to IL‐1β plus IL‐23, whereas under the same conditions it inhibited both T‐bet expression and Th1 development. These data suggest that TGF‐β is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.