Decreased GATA3 levels cause changed mouse cutaneous innate lymphoid cell fate, facilitating hair follicle recycling.

In mice, skin-resident type 2 innate lymphoid cells (ILC2s) exhibit some ILC3-like characteristics. However, the underlying mechanism remains elusive. Here, we observed lower expression of the ILC2 master regulator GATA3 specifically in cutaneous ILC2s (cILC2s) compared with canonical ILC2s, in line with its functionally divergent role in transcriptional control in cILC2s. Decreased levels of GATA3 enabled the expansion of RORγt fate-mapped (RORγtfm+) cILC2s after postnatal days, displaying certain similarities to ILC3s. Single-cell trajectory analysis showed a sequential promotion of the RORγtfm+ cILC2 divergency by RORγt and GATA3. Notably, during hair follicle recycling, these RORγtfm+ cILC2s accumulated around the hair follicle dermal papilla (DP) region to facilitate the process. Mechanistically, we found that GATA3-mediated integrin α3ß1 upregulation on RORγtfm+ cILC2s was required for their positioning around the DP. Overall, our study demonstrates a distinct regulatory role of GATA3 in cILC2s, particularly in promoting the divergence of RORγtfm+ cILC2s to facilitate hair follicle recycling.

Regulation of T helper cell differentiation by the interplay between histone modification and chromatin interaction.

The development and function of the immune system are controlled by temporospatial gene expression programs, which are regulated by cis-regulatory elements, chromatin structure, and trans-acting factors. In this study, we cataloged the dynamic histone modifications and chromatin interactions at regulatory regions during T helper (Th) cell differentiation. Our data revealed that the H3K4me1 landscape established by MLL4 in naive CD4+ T cells is critical for restructuring the regulatory interaction network and orchestrating gene expression during the early phase of Th differentiation. GATA3 plays a crucial role in further configuring H3K4me1 modification and the chromatin interaction network during Th2 differentiation. Furthermore, we demonstrated that HSS3-anchored chromatin loops function to restrict the activity of the Th2 locus control region (LCR), thus coordinating the expression of Th2 cytokines. Our results provide insights into the mechanisms of how the interplay between histone modifications, chromatin looping, and trans-acting factors contributes to the differentiation of Th cells.

GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression.

T-bet-expressing Th17 (T-bet+RORγt+) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo. Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2 Cre and Tbx21 Cre, respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via Cre-ERT2 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis of Gata3-sufficient and Gata3-deficient CNS-infiltrating CD4+ effector T cells from mixed congenic co-transfer recipient mice revealed an important, cell-intrinsic, function of GATA3 in regulating the expression of Egr2, Bhlhe40, and Csf2. Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression.

HTR2A agonists play a therapeutic role by restricting ILC2 activation in papain-induced lung inflammation.

Group 2 innate lymphoid cells (ILC2s) are a category of heterogeneous cells that produce the cytokines IL-5 and IL-13, which mediate the type 2 immune response. However, specific drug targets on lung ILC2s have rarely been reported. Previous studies have shown that type 2 cytokines, such as IL-5 and IL-13, are related to depression. Here, we demonstrated the negative correlation between the depression-associated monoamine neurotransmitter serotonin and secretion of the cytokines IL-5 and IL-13 by ILC2s in individuals with depression. Interestingly, serotonin ameliorates papain-induced lung inflammation by suppressing ILC2 activation. Our data showed that the serotonin receptor HTR2A was highly expressed on ILC2s from mouse lungs and human PBMCs. Furthermore, an HTR2A selective agonist (DOI) impaired ILC2 activation and alleviated the type 2 immune response in vivo and in vitro. Mice with ILC2-specific depletion of HTR2A (Il5cre/+·Htr2aflox/flox mice) abolished the DOI-mediated inhibition of ILC2s in a papain-induced mouse model of inflammation. In conclusion, serotonin and DOI could restrict the type 2 lung immune response, indicating a potential treatment strategy for type 2 lung inflammation by targeting HTR2A on ST2+ ILC2s.

Orchestration between ILC2s and Th2 cells in shaping type 2 immune responses.

The type 2 immune response is critical for host defense against large parasites such as helminths. On the other hand, dysregulation of the type 2 immune response may cause immunopathological conditions, including asthma, atopic dermatitis, rhinitis, and anaphylaxis. Thus, a balanced type 2 immune response must be achieved to mount effective protection against invading pathogens while avoiding immunopathology. The classical model of type 2 immunity mainly involves the differentiation of type 2 T helper (Th2) cells and the production of distinct type 2 cytokines, including interleukin-4 (IL-4), IL-5, and IL-13. Group 2 innate lymphoid cells (ILC2s) were recently recognized as another important source of type 2 cytokines. Although eosinophils, mast cells, and basophils can also express type 2 cytokines and participate in type 2 immune responses to various degrees, the production of type 2 cytokines by the lymphoid lineages, Th2 cells, and ILC2s in particular is the central event during the type 2 immune response. In this review, we discuss recent advances in our understanding of how ILC2s and Th2 cells orchestrate type 2 immune responses through direct and indirect interactions.

The transcription factor Bhlhe40 is a switch of inflammatory versus antiinflammatory Th1 cell fate determination.

Type 1 T helper (Th1) cells play a critical role in host defense against intracellular pathogens and in autoimmune diseases by producing a key inflammatory cytokine interferon (IFN)-γ; some Th1 cells can also be antiinflammatory through producing IL-10. However, the molecular switch for regulating the differentiation of inflammatory and antiinflammatory Th1 cells is still elusive. Here, we show that Bhlhe40-deficient CD4 Th1 cells produced less IFN-γ but substantially more IL-10 than wild-type Th1 cells both in vitro and in vivo. Bhlhe40-mediated IFN-γ production was independent of transcription factor T-bet regulation. Mice with conditional deletion of Bhlhe40 in T cells succumbed to Toxoplasma gondii infection, and blockade of IL-10 signaling during infection rescued these mice from death. Thus, our results demonstrate that transcription factor Bhlhe40 is a molecular switch for determining the fate of inflammatory and antiinflammatory Th1 cells.

Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation.

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. Here, we show that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in Bcl11b-deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression in Th2 cells.

Caerulomycin A suppresses the differentiation of naïve T cells and alleviates the symptoms of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a highly detrimental autoimmune disease of the central nervous system. There is no cure for it but the treatment typically focuses on subsiding severity and recurrence of the disease. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS. It is characterized by frequent relapses due to the generation of memory T cells. Caerulomycin A (CaeA) is known to suppress the Th1 cells, Th2 cells, and Th17 cells. Interestingly, it enhances the generation of regulatory T cells (Tregs). Th1 cells and Th17 cells are known to aggravate EAE, whereas Tregs suppress the disease symptoms. Consequently, in the current study we evaluated the influence of CaeA on EAE. Intriguingly, we observed by whole body imaging that CaeA regressed the clinical symptoms of EAE. Further, there was reduction in the pool of Th1 cells, Th17 cells, and CD8 T cells. The mechanism involved in suppressing the EAE symptoms was due to the inhibition in the generation of effector and central memory T cells and induction of the expansion of Tregs. In essence, these findings implicate that CaeA may be considered as a potent future immunosuppressive drug.

Caerulomycin A inhibits Th2 cell activity: a possible role in the management of asthma.

We have recently demonstrated that Caerulomycin A induces regulatory T cells differentiation by suppressing Th1 cells activity. The role of regulatory T cells is well established in suppressing the function of Th2 cells. Th2 cells are known to inflict the induction of the activation of asthma. Consequently, in the present study, we monitored the influence of Caerulomycin A in inhibiting the activity of Th2 cells and its impact in recuperating asthma symptoms. Interestingly, we observed that Caerulomycin A significantly suppressed the differentiation of Th2 cells, as evidenced by downregulation in the GATA-3 expression. Further, decline in the levels of IL-4, IL-5 and IL-13 cytokines and IgE was noted in the animals suffering from asthma. Furthermore, we noticed substantial suppression in the inflammatory response and number of eosinophils in the lungs. In essence, this study signifies an important therapeutic role of Caerulomycin A in asthma.

Caerulomycin A suppresses immunity by inhibiting T cell activity.

BACKGROUND: Caerulomycin A (CaeA) is a known antifungal and antibiotic agent. Further, CaeA is reported to induce the expansion of regulatory T cell and prolongs the survival of skin allografts in mouse model of transplantation. In the current study, CaeA was purified and characterized from a novel species of actinomycetes, Actinoalloteichus spitiensis. The CaeA was identified for its novel immunosuppressive property by inhibiting in vitro and in vivo function of T cells. METHODS: Isolation, purification and characterization of CaeA were performed using High Performance Flash Chromatography (HPFC), NMR and mass spectrometry techniques. In vitro and in vivo T cell studies were conducted in mice using flowcytometry, ELISA and thymidine-[methyl-(3)H] incorporation. RESULTS: CaeA significantly suppressed T cell activation and IFN-γ secretion. Further, it inhibited the T cells function at G1 phase of cell cycle. No apoptosis was noticed by CaeA at a concentration responsible for inducing T cell retardation. Furthermore, the change in the function of B cells but not macrophages was observed. The CaeA as well exhibited substantial inhibitory activity in vivo. CONCLUSION: This study describes for the first time novel in vitro and in vivo immunosuppressive function of CaeA on T cells and B cells. CaeA has enough potential to act as a future immunosuppressive drug.

Caerulomycin A enhances transforming growth factor-ß (TGF-ß)-Smad3 protein signaling by suppressing interferon-γ (IFN-γ)-signal transducer and activator of transcription 1 (STAT1) protein signaling to expand regulatory T cells (Tregs).

Cytokines play a very important role in the regulation of immune homeostasis. Regulatory T cells (Tregs) responsible for the generation of peripheral tolerance are under the tight regulation of the cytokine milieu. In this study, we report a novel role of a bipyridyl compound, Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4(+) Foxp3(+) cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4(+)/IFN-γ(+) and CD4(+)/IL-17(+) cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-ß-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity.

Latency-associated protein Acr1 impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

Promiscuous peptide of 16 kDa antigen linked to Pam2Cys protects against Mycobacterium tuberculosis by evoking enduring memory T-cell response.

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.