Penetrating Traumatic Brain Injury Triggers Dysregulation of Cathepsin B Protein Levels Independent of Cysteine Protease Activity in Brain and Cerebral Spinal Fluid.

Cathepsin B (CatB), a lysosomal cysteine protease, is important to brain function and may have dual utility as a peripheral biomarker of moderate-severe traumatic brain injury (TBI). The present study determined levels of pro- and mature (mat) CatB protein as well as cysteine protease activity within the frontal cortex (FC; proximal injury site), hippocampus (HC; distal injury site), and cerebral spinal fluid (CSF) collected 1-7 days after craniotomy and penetrating ballistic-like brain injury (PBBI) in rats. Values were compared with naïve controls. Further, the utility of CatB protein as a translational biomarker was determined in CSF derived from patients with severe TBI. Craniotomy increased matCatB levels in the FC and HC, and led to elevation of HC activity at day 7. PBBI caused an even greater elevation in matCatB within the FC and HC within 3-7 days. After PBBI, cysteine protease activity peaked at 3 days in the FC and was elevated at 1 day and 7 days, but not 3 days, in the HC. In rat CSF, proCatB, matCatB, and cysteine protease activity peaked at 3 days after craniotomy and PBBI. Addition of CA-074, a CatB-specific inhibitor, confirmed that protease activity was due to active matCatB in rat brain tissues and CSF at all time-points. In patients, CatB protein was detectable from 6 h through 10 days after TBI. Notably, CatB levels were significantly higher in CSF collected within 3 days after TBI compared with non-TBI controls. Collectively, this work indicates that CatB and its cysteine protease activity may serve as collective molecular signatures of TBI progression that differentially vary within both proximal and distal brain regions. CatB and its protease activity may have utility as a surrogate, translational biomarker of acute-subacute TBI.

The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+) T cells.

The molecular mechanisms by which signaling via transforming growth factor-ß (TGF-ß) and interleukin 4 (IL-4) control the differentiation of CD4(+) IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4(+) T cells. Mechanistically, TGF-ß1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4(+) T cells in vitro. Thus, our study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 differentiation.

Antibiotics in neonatal life increase murine susceptibility to experimental psoriasis.

Psoriasis is an inflammatory skin disease affecting ∼2% of the world's population, but the aetiology remains incompletely understood. Recently, microbiota have been shown to differentially regulate the development of autoimmune diseases, but their influence on psoriasis is incompletely understood. We show here that adult mice treated with antibiotics that target Gram-negative and Gram-positive bacteria develop ameliorated psoriasiform dermatitis induced by imiquimod, with decreased pro-inflammatory IL-17- and IL-22-producing T cells. Surprisingly, mice treated neonatally with these antibiotics develop exacerbated psoriasis induced by imiquimod or recombinant IL-23 injection when challenged as adults, with increased IL-22-producing γδ(+) T cells. 16S rRNA gene compositional analysis reveals that neonatal antibiotic-treatment dysregulates gut and skin microbiota in adults, which is associated with increased susceptibility to experimental psoriasis. This link between neonatal antibiotic-mediated imbalance in microbiota and development of experimental psoriasis provides precedence for further investigation of its specific aetiology as it relates to human psoriasis.

In vivo-generated antigen-specific regulatory T cells treat autoimmunity without compromising antibacterial immune response.

Harnessing regulatory T (Treg) cells is a promising approach for treating autoimmune disease. However, inducing antigen-specific Treg cells that target inflammatory immune cells without compromising beneficial immune responses has remained an unmet challenge. We developed a pathway to generate autoantigen-specific Treg cells in vivo, which showed therapeutic effects on experimental autoimmune encephalomyelitis and nonobese diabetes in mice. Specifically, we induced apoptosis of immune cells by systemic sublethal irradiation or depleted B and CD8(+) T cells with specific antibodies and then administered autoantigenic peptides in mice with established autoimmune diseases. We demonstrated mechanistically that apoptotic cells triggered professional phagocytes to produce transforming growth factor-ß, under which the autoantigenic peptides directed naïve CD4(+) T cells to differentiate into Foxp3(+) Treg cells instead of into T effector cells in vivo. These antigen-specific Treg cells specifically ameliorated autoimmunity without compromising immune responses to bacterial antigen. We have thus successfully generated antigen-specific Treg cells with therapeutic activity toward autoimmunity. The findings may lead to the development of antigen-specific Treg cell-mediated immunotherapy for multiple sclerosis and type 1 diabetes and also other autoimmune diseases.

Thymocyte apoptosis drives the intrathymic generation of regulatory T cells.

Maintenance of immune tolerance critically depends upon regulatory T cells that express the transcription factor forkhead box P3 (Foxp3). These CD4(+) T cells can be generated in the thymus, termed thymus-derived regulatory T cells (tTregs), but their developmental pathway remains incompletely understood. tTreg development has been shown to be delayed compared with that of CD4(+) single positive (SP) thymocytes, with tTregs being detected only in neonatal thymi by day 3 after birth. Here, we outline the reasons for this delayed emergence of Foxp3(+) tTregs and demonstrate that thymocyte apoptosis is intrinsically tied to tTreg development. We show that thymic apoptosis leads to the production of TGFß intrathymically from thymic macrophages, dendritic cells, and epithelial cells. This TGFß then induces foxp3 expression and drives tTreg generation. Thymocyte apoptosis has previously been shown to accelerate after birth, which drives increases in TGFß in the neonatal thymus. We highlight a paucity of TGFß in the neonatal thymus, accounting for the delayed development of tTregs compared with CD4(+) SP thymocytes. Importantly, we show that enhanced levels of apoptosis in the thymus result in an augmented tTreg population and, moreover, that decreasing thymic apoptosis results in reduced tTregs. In addition to this, we also show that T-cell receptor (TCR) signals of different affinity were all capable of driving tTreg development; however, to achieve this TGFß signals must also be received concomitant with the TCR signal. Collectively, our results indicate that thymic apoptosis is a key event in tTreg generation and reveal a previously unrecognized apoptosis-TGFß-Foxp3 axis that mediates the development of tTregs.

PARP-1 regulates expression of TGF-ß receptors in T cells.

Transforming growth factor-ß (TGF-ß) receptors (TßRs) are essential components for TGF-ß signal transduction in T cells, yet the mechanisms by which the receptors are regulated remain poorly understood. We show here that Poly(ADP-ribose) polymerase-1 (PARP-1) regulates TGF-ß receptor I (TßRI) and II (TßRII) expression in CD4(+) T cells and subsequently affects Smad2/3-mediated TGF-ß signal transduction. Inhibition of PARP-1 led to the upregulation of both TßRI and TßRII, yet the underlying molecular mechanisms were distinct. PARP-1 selectively bound to the promoter of TßRII, whereas the enzymatic activity of PARP-1 was responsible for the inhibition of TßRI expression. Importantly, inhibition of PARP-1 also enhanced expression of TßRs in human CD4(+) T cells. Thus, PARP-1 regulates TßR expression and TGF-ß signaling in T cells.

PARP-1 controls immunosuppressive function of regulatory T cells by destabilizing Foxp3.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme and transcription factor that is involved in inflammatory response, but its role in T cell response remains largely unknown. We show here that PARP-1 regulates the suppressive function of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Specifically, Tregs in mice with a null mutation of the PARP-1 gene (PARP-1(-/-)) showed significantly stronger suppressive activity than did wild-type Tregs in culture. We elucidate that this enhanced suppressive function is attributed to sustained higher expression of Foxp3 and CD25 in PARP-1(-/-) Tregs. Furthermore, in PARP-1(-/-) Tregs, Foxp3 protein shows substantially higher levels of binding to the conserved non-coding DNA sequence 2 (CNS2) at the foxp3 gene, a region important in maintaining Foxp3 gene expression in Tregs. Thus, our data reveal a role for PARP-1 in controlling the function of Tregs through modulation of the stable expression of Foxp3.