TGF-β1 enhances mouse mast cell release of IL-6 and IL-13

For immune cells, TGF-β1 can enhance or repress effector functions. Here, we characterize the effects of TGF-β1 on IgE-mediated activation of primary murine mast cells derived from hematopoietic stem cells (BMMC). We also investigated potential interaction between TGF-β1 and stem-cell factor (SCF). Resting IL-6 production was increased with TGF-β1 but significance was lost following BMMC activation via IgE receptor (FcεRI) crosslinking. SCF also enhanced resting levels of IL-6, but there was no difference from control once FcεRI was engaged. SCF had no effect on IL-13 production; however, TGF-β1 treatment enhanced release of IL-13 upon FcεRI activation. Lastly, percent colocalization of SCF receptor (CD117) and FcεRI were unaffected by TGF-β1 treatment. These data reveal a novel positive effect of soluble TGF-β1 on mast cell activation.


Introduction 19
Transforming Growth Factor Beta (TGF-b1) is a widely expressed cytokine. The TGF-β1 signaling 20 pathway evolved approximately one billion years ago as an immune regulatory mechanism among 21 vertebrates. TGF-β1 modulates cellular responses starting with binding to TGF-b receptor II (TGF-βRII). 22 TGF-βRII then aggregates with TGF-βRI at the cell surface inducing the phosphorylation of Smad proteins 23 intracellularly. Ultimately, this cascade reaches the nucleus for transcriptional regulation. TGF-β1 is 24 documented as generally inhibitory starting in the 1990s: as an anti-inflammatory, anti-autoimmune 25 cytokine [1]. 26 Mast cells are myeloid lineage cells of hematopoietic origin. They are present in the skin and along 27 mucosal membranes, especially the gut where they combat helminth parasites. Well known for their 28 roles in allergic pathologies, mast cells also are key to physiology at mucosal barriers. Mast cells are 29 capable of collecting and presenting antigen to other cells [2]. They are also central in driving a T H 2 30 response inducing B cells to class switch to IgE via IL-4 and IL-13 [3]. Canonical activation of mast cells 31 starts with the priming of their high affinity IgE receptor, FcεRI. In the body, mast cells are stably coated 32 with IgE bound to FcεRI. Upon multivalent antigen binding of these IgE-FcεRI complexes, the receptors 33 cluster -crosslink, and internalize, triggering signaling cascades resulting in degranulation of the cell and 34 activation of transcription factors, such as STAT5, to upregulate cytokine production [3]. Hours to days 35 later these cytokines (e.g., IL-6, IL-13) are secreted [2] . They also exhibit an alternative pathway to 36 activation through IL-33 and its receptor (ST2), independent of FcεRI [4]. 37 Recent evidence suggests that the interaction between mast cells and typical immunosuppressive 38 cytokines varies from other immune cells. A typical inhibitory cytokine, IL-10, was shown to behave as 39 an immunostimulant when given to mast cells and in the development of mucosal food allergies [5]. At 40 the post-transcriptional level, IL-10 was found to regulate microRNAs, which enhanced skin mast cell 41 secretion of IL-6 and IL-13 [6]. These interactions necessitate closer examination of the behavior of mast 42 cells regarding common stimulatory and inhibitory molecules. Herein, we describe effects of TGF-β1 on 43 IgE-mediated mast cell activation by observing the production of two cytokines commonly associated 44 with this process: IL-6 (an inflammatory T H -1/T H -17 cytokine) and IL-13 (a chemotactic cytokine that 45 drives a T H -2 response). We demonstrate that soluble TGF-β1 amplifies mast cell release of these 46 cytokines. This effect opposes SCF exposure, depending on the cytokine; and it is unlikely due to 47 receptor interference at the membrane. were maintained between 250,000 and 1,000,000 cells/mL for 4-6 weeks to achieve pure BMMCs. 62 All cultures were randomly divided into SCF-treated and untreated groups during differentiation. SCF-63 treated cell populations were given 10ng/mL SCF (Peprotech). 64

TGF-β1 Treatment 65
For TGF-β1 treated groups, SCF-treated and untreated cells were distributed in 6-well plates at a density 66 between 250,000 and 1,000,000 cells/mL. Cells were treated with 2ng/mL murine TGF-β1 (Cell 67 Signaling) and maintained for two days before IgE priming. 68

Statistics 89
Cytokine secretion was measured in pg/mL per 10 6 cells. Cytokine levels were also quantified based on 90 the fold change compared to the untreated controls. Data was tested for normality using the Shapiro-91 Wilks test and transformed accordingly. 92 Cytokine release data were analyzed via Welch's t-test. Significance was noted at p≤0.05. 93 Fluoresence microscopy data are reported as least square means±standard error of the mean with 94 significance at p<0.05. The effects of TGF-β1 and FceRI crosslinking on colocalization of BMMC surface 95 receptors were analyzed using one-way analysis of variance. 96

TGF-β1 enhances IL-6 secretion by BMMCs 98
BMMCs were treated with TGF-β1, SCF, or both, and then activated by crosslinking FcεRI. The following 99 IL-6 secretion data were collected from at least 4 separate BMMC populations (i.e., bone marrow 100 differentiated from different mice). IL-6 was increased following IgE-mediated activation (Figure 1 a), 101 but IL-6 was also increased in cells treated with either SCF or TGF-β1 alone independent of IgE-mediated activation; cells treated with both ligands exhibited higher levels compared to untreated controls ( Figure  103 1 c). When cross-linked, only TGF-β1 appeared to enhance IL-6 secretion but the difference was not 104 significant (Data not shown). These data show that soluble TGF-β1 directly enhances mast cell IL-6 105 production independent of IgE-mediated activation. 106

TGF-β1, but not SCF, enhances IL-13 secretion from BMMCs. 107
Concentrations of IL-13 were also measured as the fold change when crosslinked. Un-crosslinked 108 populations secreted little to no detectable IL-13. Figure 1 b shows the raw IL-13 production based on 109 treatment. Unlike IL-6, IL-13 production prior to IgE activation was not affected based on cytokine 110 treatment. However, treatment with TGF-β1 resulted in higher levels of IL-13 upon IgE-mediated 111 activation -this effect persisted when co-treated with SCF, but not with SCF alone (Figure 1 d). These 112 data show that soluble TGF-β1 enhances IL-13 production upon FceRI engagement. 113

Discussion 120
In our experiments soluble TGF-β1 stimulates IL-6 secretion independent of IgE-mediated activation. 121 TGF-β1 has been shown to promote mast cell IL-6 production in the context of lung inflammation; this 122 promotes neutrophil apoptosis and clearance [7]. However, this mechanism involved T reg cell surface 123 sequestered TGF-β1 [8]. Our findings suggest that TGF-β1 plays a directly stimulatory role on IL-6 124 production, potentially independent of immunosuppressive T reg , which could elicit acute phase 125 inflammatory responses. Characterizing the mechanism(s) involved and carefully testing the direct effect(s) of soluble TGF-β1 on other myeloid cells is warranted. Alternatively, myeloid-derived 127 suppressor cells (MDSC) utilize IL-6 to support tumor progression [9]. MDSCs also enhance IL-6 and IL-128 13 secretion by activated mast cells [10]. The careful study of interactions between TGF-β1, IL-6, T reg , 129 and mast cells will be insightful in chronic inflammatory settings, such as high-grade solid cancers. 130 Here, TGF-β1 also enhances production of IL-13 following mast cell activation via FceRI. IL-13 is a major 131 cytokine in T H 2-related immune responses, likey responsible for the clearing of large extracellular 132 insults, such as gut parasites [11]. However, unmitigated IL-13 release will drive B-lymphocyte class 133 switching to IgE, which in turn coats naïve mast cells via FceRI, thus prompting a vicious T H 2 cycle [12]. 134 Our experiments show that TGF-β1 directly enhances IL-13 production, which propagates such 135 pathologies. 136 Through fluorescence microscopy we observed any effects TGF-β1, and its receptor TGFBRII, might have 137 at the signaling apex of the definitive mast cell surface receptors FceRI and CD117 (SCF receptor). FceRI 138 and CD117 colocalization is lost upon crosslinking of FceRI with IgE-antigen complexes. This is not 139 surprising as FceRI internalization after cross-linking is known [13]. However, this decline in 140 colocalization is unchanged with TGF-β1 treatment, suggesting there is no apparent surface cross-talk 141 occurring among these receptors. It has been shown that TGF-β1 transcriptionally represses FcεRI and 142 CD117 through regulation of Etf homologous factor (Ehf) [14]. This inhibition was not observed in our 143 studies, suggesting reduced receptor expression of CD117 through flow cytometric analysis does not 144 directly correspond to reduced receptor expression. 145 We demonstrate that TGF-β1 plays a direct stimulatory role on primary mast cells in vitro. The specific 146 molecular mechanism underlying this effect is still unknown. TGF-β1 might modulate targets outside of 147 the canonical TGF-β1 signaling cascade (Smads) as preliminary experiments using a TGF-βR1 inhibitor 148 resulted in no significant differences in cytokine expression (data not shown). IL-6 secretion was 149 increased independent of IgE-mediated activation, suggesting that TGF-β1 non-canonically targets the MAP kinase or Akt pathways to enhance IL-6 production [15]. Future research into the phosphorylation 151 patterns of this pathway will reveal the specific effects of TGF-β1. Additionally, IL-13 production was 152 altered only after IgE activation, suggesting that TGF-β1 modulates the FcεRI signaling pathway (e.g., 153 STAT5). Altogether these data necessitate careful examination of soluble TGF-β1 with respect to mast 154 cell effector functions. 155

Authors' Contributions 156
DL undertook experiments, design and data analysis, and drafted the manuscript; MP performed 157 microscopic analyses; NP conceived, designed, and coordinated the study, and assisted manuscript 158 drafting. All authors gave final approval for publication. 159

Competing Interests 160
The authors declare no competing financial interests. 161