You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


T~H~17 cells are a selective lineage of CD4^+^ T helper cells that are critical for host defense and autoimmunity by expressing the proinflammatory cytokines IL-17A, IL-17F, and IL-22 ([@bib17]; [@bib19]; [@bib16]). The induction of T~H~17 cells during inflammatory conditions such as experimental autoimmune encephalomyelitis (EAE) requires cytokines such as IL-1β, IL-6, IL-23, and TGF-β1 ([@bib16]). In addition to their presence during inflammatory responses, a population of T cells that expresses retinoic acid receptor--related orphan receptor γt (Rorγt), which is a T~H~17-specific transcription factor, is also found at steady state (sT~H~17) in the small intestine lamina propria (LP; [@bib12]; [@bib13]), where they accumulate only in the presence of luminal commensal microbiota ([@bib1]; [@bib9]; [@bib13]). Recently, one member of this bacterial community, the *Clostridia*-related segmented filamentous bacteria (SFB), was shown to be capable of inducing the generation of T~H~17 cells, as well as other subsets of T~H~ cells ([@bib14]). It has been suggested that the induction of ATP ([@bib1]) and serum amyloid proteins ([@bib14]) by commensal bacteria are required for promoting the generation of intestinal T~H~17 cells. However, the host signaling pathway linking the microbiota to the induction of intestinal sT~H~17 cells at steady state remains to be fully elucidated.

IL-1R signaling is important for murine T~H~17 development ([@bib23]; [@bib2]; [@bib8]) and accordingly, mice lacking IL-1R expression exhibit impaired capacity to generate T~H~17 cells in the setting of EAE ([@bib21]; [@bib2]). Furthermore, the IL-1β--IL-1R signaling pathway has been shown to promote IL-17 production by the unconventional γδ T cells in the presence of commensal bacteria ([@bib22]; [@bib5]). Based on analysis of intracellular staining of LP cells for T~H~17-associated cytokines after stimulation with phorbol ester and ionomycin, it was suggested that myeloid differentiation factor 88 (MyD88) signaling pathways that include Toll-like receptor (TLR) and the IL-1β--IL-1R signaling pathways were not important in inducing the development of normal T~H~17 cell responses in the small intestine ([@bib1]; [@bib14]). Because recent findings revealed that phorbol ester and ionomycin stimulation may exaggerate intracellular IL-17 expression in T~H~17 cells ([@bib11]), it remains unclear whether MyD88 signaling pathways play a role in the induction of intestinal sT~H~17 cells in animals colonized with gut-residing bacteria. In the present study, we examined the role of IL-1R and MyD88 signaling in influencing the induction of sT~H~17 cells in the intestinal microenvironment. Our results revealed that the microbiota induces the production of IL-1β, but not IL-6, in LP phagocytes and that stimulation of IL-1β--IL-1R signaling is necessary and sufficient in driving the generation of intestinal sT~H~17 cells.

RESULTS AND DISCUSSION
======================

IL-1β--IL-1R signaling promotes the development of intestinal sT~H~17 cells
---------------------------------------------------------------------------

To assess the role of IL-1R signaling in the development of sT~H~17 cells under steady-state conditions in vivo, we crossed *Rorc(γt)-gfp* reporter mice, which express GFP under the control of the promoter of Rorγt ([@bib6]; [@bib12]), with *Ilr1*^−/−^ mice to generate *Rorc(γt)-gfp* reporter mice in the presence and absence of IL-1R. Although the frequency of total CD4^+^ T cells was not significantly different ([Fig. 1 A](#fig1){ref-type="fig"}), the numbers of Rorc(γt)-gfp^+^ CD4^+^ T cells were greatly reduced in the LP of *Ilr1*^−/−^ mice compared with those in wild-type littermates ([Fig. 1, B and C](#fig1){ref-type="fig"}). The impaired generation of LP sT~H~17 cells was not the result of enhanced development of Foxp3^+^ T regulatory cells in the absence IL-1R1 signaling, given that no differences were observed in the frequency of Foxp3^+^ CD4^+^ T cells between *Ilr1*^−/−^ and the littermate control mice ([Fig. 1 D](#fig1){ref-type="fig"}). Consistent with the reduced frequency of sT~H~17 cells in vivo, the production of T~H~17-associated effector cytokines (IL-17 and IL-22) by FACS-sorted intestinal CD3^+^CD4^+^ IL-1R^−/−^ cells, was also blunted after in vitro stimulation ([Fig. 1 E](#fig1){ref-type="fig"}). The impaired generation of sT~H~17 cells observed in the *Ilr1*^−/−^ mice was not caused by the lack of intestinal colonization with SFB, as *Ilr1*^−/−^ and controls animals maintained in our specific pathogen--free (SPF) facility had a comparable number of fecal SFB as determined by quantitative real-time PCR (qPCR) of 16s rRNA gene sequences (unpublished data). In addition, the expression of *IL-6* and *IL-23p19* in the small intestine was unaffected by the absence of IL-1R1 signaling, as qPCR analysis revealed that the transcript levels of these genes were similar between *Ilr1*^−/−^ and wild-type littermate animals (unpublished data). The proinflammatory cytokine IL-1β is a known ligand that interacts with IL-1R1 to mediate its biological effects ([@bib20]). Accordingly, the in vitro production of IL-17 and IL-22 by LP cells isolated from *Il1β^−/−^* and *Ilr1*^−/−^ mice was similarly reduced ([Fig. 1 F](#fig1){ref-type="fig"}). In contrast, the production of the Th1-associated cytokine IFN-γ by total LP cells was not impaired after in vitro stimulation (unpublished data).

![**IL-1β--IL-1R and MyD88 signaling promotes the development of intestinal sT~H~17 cells**. (A--C) Representative FACS dot plots of small intestine LP lymphoid cells from the indicated mice are shown in A and B, and the percentage of Rorγt-gfp^+^ CD3^+^CD4^+^ of individual mice are shown in C. Cells were pregated on total lymphocyte population in A and on CD3^+^ cells in B. (D) Representative FACS dot plot analysis of Foxp3 expression gated on total LP CD3^+^CD4^+^ cells of indicated mice. (E) Production of intestinal IL-17 and IL-22 by sorted CD3^+^CD4^+^ cells from the indicated mice was assessed ex vivo after stimulation with anti-CD3. (F) Production of IL-17 and IL-22 by (10^6^ cells/ml) total LP cells from indicated mice was assessed ex vivo after stimulation with anti-CD3. (G) Production of intestinal IL-17 and IL-22 by sorted CD3^+^CD4^+^ cells from the indicated mice was assessed ex vivo after stimulation with anti-CD3. (H) Representative FACS dot plots gated on small intestine LP CD3^+^ cells in the indicated mice. (I) Production of intestinal IL-17 and IL-22 by sorted CD3^+^CD4^+^ cells from the indicated mice was assessed ex vivo after stimulation with anti-CD3 antibody (top). Representative FACS dot plots gated on small intestine LP CD4^+^ cells in the indicated mice (bottom). In vitro cytokine results shown in panels E--G and I are presented as means + SD from three to four individual mice per group. FACS results shown in panels A, B, D, H, and I are representative of two to five independent experiments with similar results.](JEM_20111703_Fig1){#fig1}

The development of intestinal sT~H~17 responses requires MyD88, but not IL-6
----------------------------------------------------------------------------

Given that the adaptor molecule MyD88 plays a critical role in both TLR- and IL-1R--signaling pathways ([@bib18]), we reevaluated the intestinal sT~H~17 response in *Myd88^−/−^* mice. Consistent with previous studies ([@bib1]; [@bib13]), we observed normal intestinal T~H~17 responses in the absence of MyD88 signaling by intracellular cytokine staining of LP cells stimulated with phorbol ester and ionomycin (unpublished data). However, in light of recent findings suggesting that phorbol ester and ionomycin stimulation may exaggerate intracellular IL-17 expression in T~H~17 cells ([@bib11]), we sought to address the role of MyD88 in promoting T~H~17 responses under more physiological conditions. To do so, we stimulated FACS-sorted LP CD3^+^CD4^+^ T cells isolated from *Myd88^−/−^* animals with antibody to CD3. *Myd88^−/−^* mice exhibited decreased intestinal T~H~17 cytokine responses as determined by ex vivo stimulation of CD3^+^CD4^+^-purified cells ([Fig. 1 G](#fig1){ref-type="fig"}). To further assess the role of MyD88 in promoting sT~H~17 responses without ex vivo manipulation, we crossed *Rorc(γt)-gfp* reporter mice with *Myd88^−/−^* mice to generate *Rorc(γt)-gfp* reporter mice. Consistent with the results obtained with *Ilr1*^−/−^ mice in vivo and stimulation of LP CD3^+^CD4^+^ T cells with anti-CD3 ex vivo, we found reduced frequency of CD4^+^ *Rorc(γt)-gfp^+^* cells in the intestinal LP of *Myd88^−/−^Rorc(γt)-gfp* mice when compared with *Myd88^+/−^Rorc(γt)-gfp* littermate mice ([Fig. 1 H](#fig1){ref-type="fig"}). In contrast, the frequency of CD4^+^ *Rorc(γt)-gfp^+^* cells in the intestinal LP of *IL-6^−/−^Rorc(γt)-gfp* mice was unimpaired when compared with *IL-6^+/−^Rorc(γt)-gfp* littermate mice ([Fig. 1 I](#fig1){ref-type="fig"}). Consistently, the production of T~H~17-associated effector cytokines (IL-17 and IL-22) by FACS-sorted intestinal CD3^+^CD4^+^ IL6^−/−^ cells was comparable to that of intestinal CD3^+^CD4^+^ T cells from *IL-6^+/−^* littermate mice ([Fig. 1 I](#fig1){ref-type="fig"} and not depicted). These observations indicate that the homeostatic development of intestinal sT~H~17 cells does not require IL-6, but is dependent on, IL-1β--IL-1R and MyD88 signaling.

The CD11b^+^F4/80^+^CD11c^−/low^-resident macrophage population is the main source of IL-1β in the small intestine
------------------------------------------------------------------------------------------------------------------

We next investigated which cellular subset of cells in the intestine is a potential source of IL-1β. To begin to address this question, we generated bone marrow chimera mice by reconstituting lethally irradiated wild-type recipient mice with bone marrow from wild-type or IL-1β--deficient mice. Analysis of the chimera mice revealed that IL-1β produced by hematopoietic cells is required for the induction of intestinal sT~H~17 responses ([Fig. 2 A](#fig2){ref-type="fig"}). Thus, deficiency of IL-1β in hematopoietic cells impaired the production of the T~H~17-associated effector cytokines, IL-17, and IL-22, but not IFN-γ, by LP cells after in vitro stimulation ([Fig. 2 A](#fig2){ref-type="fig"}). Accumulating evidence suggests that LP mononuclear phagocytes are crucial for maintaining intestinal homeostasis and promoting intestinal immunity ([@bib24]). Accordingly, in the intestinal LP, we identified, based on the differential surface expression of CD11c and CD11b, three distinct mononuclear phagocyte populations: CD11b^+^CD11c^−^ (R1), CD11b^+^CD11c^+^ (R2), and CD11b^−^CD11c^+^ (R3; [Fig. 2 B](#fig2){ref-type="fig"}, left). Next, we examined which subsets of these intestinal phagocytes contribute to steady-state production of IL-1β. To do so, we sorted R1, R2, and R3 populations and assessed intracellular IL-1β protein levels. The R1 population of cells expressed the highest level of IL-1β protein compared to the R2 or R3 subsets ([Fig. 2 B](#fig2){ref-type="fig"}, middle). Further flow cytometry analysis demonstrated that the R1 population was CD11b^+^, F4/80^+^, MHCII^+^, CD11c^−/low^, and CD103^int^ (unpublished data); thus, this population seems to belong to a previously described subset of CD11b^+^F4/80^+^ intestinal macrophages ([@bib15]; [@bib3]). Therefore, these findings indicate that the CD11b^+^-, F4/80^+^-, MHCII^+^-, CD11c^−/low^-, and CD103^int^-resident populations are the main source of IL-1β production in the LP of the small intestine.

![**The microbiota promotes MyD88-mediated signaling for IL-1β induction in resident intestinal macrophages.** (A) Bone marrow chimeras were generated by reconstituting lethally irradiating SPF wild-type recipients with 10^6^ donor bone marrow cells isolated from the indicated mice. Intestinal sT~H~17 response was assessed at 10 wk after reconstitution. Production of IL-17, IL-22, and IFN-γ by (10^6^ cells/ml) total LP cells was assessed in the indicated chimera mice after ex vivo stimulation with anti-CD3. The results are shown from one of two independent experiments with five chimera mice per group per experiment. (B, left) Representative FACS dot plot analysis of total intestine LP cells based on CD11b and CD11c expression. (B, middle) R1, R2, and R3 populations were FACS sorted from SPF wild-type mice, and IL-1β levels in total cell lysates from the indicated populations were determined by ELISA and normalized to total protein concentration. (B, right) R1, R2, and R3 subsets were sorted from the LP of the indicated mice, and IL-1β levels were determined. (C) IL-1β mRNA levels measured by real-time RT-PCR in sorted R1 population from indicated mice. (D) Protein extracts form sorted intestinal R1 population from indicated mice were immunoblotted with anti--IL-1β or anti--β-actin (loading control). (E) Total LP cells were isolated from either SPF (*n* = 20) or GF (*n* = 9) wild-type mice and the level of spontaneous IL-6 production in overnight cultured supernatant was determined by ELISA. Representative results are shown from one of at least two to three independent experiments. Experiments in panels C and D are representative of two experiments using pooled cells from *n* = 2--3 mice.](JEM_20111703_Fig2){#fig2}

IL-1β induction in intestinal macrophages is mediated via MyD88, but not IL-1R, signaling
-----------------------------------------------------------------------------------------

The expression of IL-1β is induced by TLR ligands and IL-1β itself can also serve as a positive feedback regulator of this pathway ([@bib4]). To investigate the interaction between commensal bacteria and the host immune system that promotes the induction of IL-1β, R1, R2, and R3 subsets from the LP of wild-type and *Ilr1*^−/−^ mice were sorted and the protein level of IL-1β was determined. There was no detectable difference in the level of IL-1β between wild-type and *Ilr1*^−/−^ animals ([Fig. 2 B](#fig2){ref-type="fig"}, right), indicating that IL-1β--IL-1R signaling does not regulate the expression of IL-1β in intestinal macrophages. This suggests that induction of IL-1β is upstream of IL-1R signaling. However, LP CD11b^+^CD11c^−^ cells from SPF *Myd88^−/−^* and germ-free (GF) wild-type mice showed decreased levels of pro--IL-1β ([Fig. 2 C](#fig2){ref-type="fig"}), suggesting that the interaction between TLR/MyD88 and the commensal microbiota is an essential requirement for the induction of IL-1β in phagocytic cells. In line with this notion, the levels of IL-1β mRNA in the LP were similarly decreased in GF wild-type and SPF *Myd88^−/−^* animals as compared with SPF control mice ([Fig. 2 D](#fig2){ref-type="fig"}). In contrast, comparable levels of IL-6 were found in the LP of wild-type and GF mice ([Fig. 2 E](#fig2){ref-type="fig"}). Collectively, these observations suggest that the microbiota regulates the production of IL-1β in LP phagocytic cells via MyD88, independent of IL-1R signaling, to promote the induction of sT~H~17 cells in the small intestine.

IL-1R signaling on T cells is required for the generation of intestinal sT~H~17 response
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IL-1R1 is ubiquitously expressed on both hematopoietic and nonhematopoietic cells ([@bib4]; [@bib20]). To assess the importance of IL-1R1 signaling in various intestinal cellular populations in promoting the generation of sT~H~17 cells, we generated bone marrow chimera mice. Reconstitution of lethally irradiated recipients, regardless of genotype, with *Ilr1*^−/−^ bone marrow recapitulated the defective sT~H~17 response observed in intact *Ilr1*^−/−^ animals ([Fig. 3 A](#fig3){ref-type="fig"}). Thus, deficiency of IL-R1 in hematopoietic cells impaired the production of the T~H~17-associated effector cytokines (IL-17 and IL-22) by LP cells after in vitro stimulation ([Fig. 3 A](#fig3){ref-type="fig"}). The requirement of an intact IL-1R1 signaling pathway in the hematopoietic compartment using bone marrow chimeric mice does not distinguish whether or not direct IL-1R1 signaling on T cells promotes the induction of intestinal sT~H~17 cells. To this end, we adoptively transferred *Ilr1*^+/−^ or *Ilr1*^−/−^ -CD3^+^CD4^+^ *Rorc(γt)-gfp*^negative^ cells into SPF *Rag1^−/−^* recipients ([Fig. 3 B](#fig3){ref-type="fig"}, left). Reconstituting *Rag1^−/−^* recipients with either *Ilr1*^+/−^ or *Ilr1*^−/−^*Rorc(γt)-gfp*^negative^ CD4^+^ T cells resulted in a similar recruitment and expansion of CD4^+^ T cells to peripheral organs and frequency of GFP-expressing CD4^+^ T cells in the spleen and liver ([Fig. 3 B](#fig3){ref-type="fig"}, middle). However, despite normal recruitment to and expansion in the intestinal LP, the absence of IL-1R1 on CD4^+^ T cells selectively impaired the ability of the donor cells to acquire Rorγt expression specifically in the intestinal microenvironment ([Fig. 3 B](#fig3){ref-type="fig"}, right). Consistent with diminished numbers of *Rorc(γt)-gfp^positive^* cells present in the intestinal LP of *Ilr1*^−/−^ T cell reconstituted *Rag1^−/−^* recipients, in vitro production of IL-17 was also negatively impacted by the lack of IL-1R1 expression on T cells ([Fig. 3 C](#fig3){ref-type="fig"}). Thus, these results emphasize that maintaining IL-1β responsiveness in T cells in the intestine is necessary for the development of sT~H~17 cells.

![**IL-1R signaling on T cells is required for the generation of intestinal sT~H~17 response**. (A) Bone marrow chimeras were generated by reconstituting lethally irradiating SPF wild-type or *Ilr1^−/−^* recipients with 10^6^ donor bone marrow cells isolated from the indicated mice. Intestinal sT~H~17 response was assessed at 10 wk after reconstitution. Production of IL-17, IL-22, and IFN-γ by (10^6^ cells/ml) total LP cells was assessed ex vivo in the indicated chimera mice after stimulation with (soluble, 1 µg/ml) anti-CD3. The results are shown from one of two independent experiments with five chimera mice per group per experiment. (B, left) Experimental procedure for adoptive T cell transfer. (B, right) Representative FACS dot plot analysis of the frequency of CD4^+^Rorγt-gfp^+^ cells in various organs at day 14 after adoptive transfer. (C) Production of IL-17 and IFN-γ by (10^6^ cells/ml) total LP cells isolated from adoptively transferred mice (B, right) after ex vivo stimulation with anti-CD3. Data shown are from one representative experiment of three independent experiments with five chimeric mice per group per experiment.](JEM_20111703_Fig3){#fig3}

Administration of IL-1β is sufficient to induce the maturation of splenic CD3^+^CD4^+^Rorγt^negative^ cells into intestinal Rorγt-expressing sT~H~17 cells in GF mice
---------------------------------------------------------------------------------------------------------------------------------------------------------------------

We next asked whether administration of IL-1β was capable of inducing the presence of sT~H~17 cells in the LP of GF animals that are largely devoid of these cells ([@bib1]; [@bib9]; [@bib13]). To assess this, GF mice were injected with rIL-1β or PBS as a control and the presence of sT~H~17 cells and production of T~H~17-associated cytokines were assessed in the small intestine. As expected, the production of T~H~17-associated cytokines IL-17 and IL-22 by LP cells was low in GF mice treated with PBS ([Fig. 4 A](#fig4){ref-type="fig"}). Importantly, IL-17 and IL-22 production by LP cells was markedly increased in GF mice treated with exogenous IL-1β when compared with mice treated with PBS ([Fig. 4 A](#fig4){ref-type="fig"}). To further interrogate the role of IL-1β in the induction of intestinal sT~H~17 response, GF wild-type animals were first infused with FACS-purified Thy1.1^+^/1.2^+^CD3^+^CD4^+^ *Rorc(γt)-gfp*^negative^ cells derived from SPF wild-type donors and then treated with PBS or exogenous rIL-1β ([Fig. 4 B](#fig4){ref-type="fig"}, top). Administration of rIL-1β selectively induced the donor CD4^+^ reporter cells to acquire *Rorc(γt)* expression in the intestinal microenvironment, but not in other peripheral organs, such as the spleen ([Fig. 4 B](#fig4){ref-type="fig"}, bottom). These data indicate that the presence of IL-1β is sufficient to induce the differentiation of intestinal sT~H~17 cells in the absence of microbiota.

![**Administration of IL-1β is sufficient to induce the maturation of splenic CD3^+^CD4^+^Rorγt^negative^ cells into intestinal Rorγt-expressing sT~H~17 cells in GF mice.** (A) GF mice were treated every other day for 14 d with either PBS or rIL-1β (1 µg/mouse), and then LP cells were isolated for analysis. Cytokine secretion by total LP cells isolated from either PBS or rIL-1β--treated animals was assessed after ex vivo stimulation with anti-CD3. (B) Experimental scheme for adoptive T cell transfer of sorted splenic Rorγt-GFP^−^CD4^+^ T cells into GF recipients. (C) Recipient GF mice were treated every other day for 14 d with either PBS (right) or rIL-1β (right), and then analyzed. Representative FACS plot gated on CD3^+^CD4^+^Thy1.1^+^/1.2^+^ donor or CD3^+^CD4^+^Thy1.1^−^/1.2^+^ recipient cells isolated from intestinal LP and spleen from the indicated recipient GF mice are shown. Analysis of Rorγt-GFP staining in recipient CD4^+^ cells is shown as a control. Representative results are shown from three independent experiments and five mice per group.](JEM_20111703_Fig4){#fig4}

The blunted intestinal sT~H~17 response of GF animals is well-characterized ([@bib13]); however, the reason for the impaired sT~H~17 response that is associated with the absence of commensal microbiota is currently unclear. We provide evidence that the microbiota induces the production of IL-1β in resident macrophages via MyD88 and IL-1β-IL-1R1 signaling is critical for the development of sT~H~17 cells in the small intestine. Administration of rIL-1β selectively induced the donor reporter cells to acquire *Rorc(γt)*-expression in the intestinal microenvironment, but not in other peripheral organs, such as the spleen. Thus, the results suggest that the IL-1β/IL-1R axis is not only required, but also sufficient, to drive the development of sT~H~17 in the intestinal microenvironment. Unexpectedly, IL-6 was not required for the development of intestinal sT~H~17 cells. Because IL-6 is important for the induction of T~H~17 cells during inflammatory conditions such as EAE ([@bib16]), the results suggest differential regulation for the development of T~H~17 cell population which may be explained by differences in the local tissue environment or specific triggers. These results do not rule out that IL-1β acts in concert with other cytokines such as IL-23 that are known to be critical for the development of T~H~17 cells during inflammatory conditions in nonintestinal tissues ([@bib16]). However, our results clearly indicate that IL-1β, but not IL-6, is induced by the microbiota and critical for the development of intestinal sT~H~17 cells in vivo.

Our findings also identified MyD88 as a critical mediator for the induction of IL-1β in intestinal macrophages while the expression of pro--IL-1β in intestinal phagocytes was not impaired in IL-R1-deficient mice. These results suggest that MyD88 acts at two distinct steps to regulate the development of sT~H~17 cells in the intestine. First, MyD88 links the microbiota to pro--IL-1β induction in intestinal macrophages. This mechanism likely involves the TLR-MyD88 signaling pathway given that this appears to be the major innate immune pathway by which the intestinal microflora stimulate the production of proinflammatory cytokines in host cells ([@bib10]). The second step involves IL-1β/IL-R1/MyD88 signaling in CD4 T cells to drive the development of intestinal sT~H~17 cells. Collectively, our data suggest that interactions between commensal bacteria and TLR signaling mediated through MyD88 promote IL-1β production in intestinal CD11b^+^ macrophages, which in turn induces the differentiation of intestinal sT~H~17 cells. Notably, exogenous IL-1β increased sT~H~17 cells in the intestine, but not in the spleen, in the absence of microbiota. We do not have an explanation for the latter results, but it suggests that the regulation of Th17 cell differentiation is more complex that originally described and varies in different tissue environments. Because T~H~17 cells have been suggested to play either a detrimental role in autoimmune disease ([@bib17]; [@bib19]; [@bib16]) or protective role during enteric infection ([@bib14]), further understanding the mechanism that regulate the quiescence and the reactivation of these cells may have therapeutic potential.

MATERIALS AND METHODS
=====================

### Animals.

SPF wild-type and *Rag-1^−/−^* mice in C57BL/6 background were originally purchased from The Jackson Laboratory. *Ilr1*^−/−^, *Il1β*^−/−^, and *Myd88^−/−^* mice in C57BL/6 background have been previously described ([@bib7]). *Il-6^−/−^* mice in C57BL/6 background were obtained from Dr. Evan Keller, the University of Michigan. *Rorc(γt)*-gfp mice were obtained from The Jackson Laboratory and crossed to *Ilr1*^−/−^, *Myd88^−/−^*, or *Il-6^−/−^* mice to generate gene-deficient reporter mice. GF wild-type mice in C57BL/6 background were bred and maintained at the Germ-Free Animal Core Facility of the University of Michigan. GF mice were maintained in flexible film isolators and were checked weekly for GF status by aerobic and anaerobic culture. The absence of microbiota was verified by microscopic analysis of stained cecal contents to detect unculturable contamination. All animal studies were performed according to approved protocols by the University of Michigan Committee on Use and Care of Animals.

### Cell isolation, adoptive transfer, and administration of IL-1β in vivo.

To isolate *Rorc(γt)-gfp^negative^* cells for adoptive transfer, pan T cells microbeads (Miltenyi Biotec) were initially used to enrich total T cells, followed by FACS sorting of CD3^+^CD4^+^*Rorc(γt)-gfp^negative^* cells. For adoptive transfer of sorted CD3^+^CD4^+^*Rorc(γt)-gfp^negative^* cells into intact GF animals, wild-type Thy1.2^+^/1.2^+^ R*orc(γt)-gfp* were crossed to Thy1.1 mice (The Jackson Laboratory) and the F1 progeny were used as donors. Donor CD4 cells were distinguished from recipient CD4 cells by flow cytometric analysis for the co-expression of Thy1.1 and Thy1.2 markers. GF mice were treated every other day for 14 d with either PBS or 1 µg/mouse of rIL-1β (PeproTech) i.p., and then intestinal tissue was harvested for analyses. After the last round of injections, there was no evidence of bacterial contamination in the rIL-1β as determined by standard culture conditions.

### Isolation and stimulation of LP cells.

Mice were killed and intestines were removed and placed in ice-cold HBSS + 1% heat inactivated FBS + 0.1% Pen/Strep (complete HBSS). After removal of residual mesenteric fat tissue, Peyer's patches were carefully excised and the intestine was flushed two times with 10 ml complete HBSS. After flushing, the intestine was opened longitudinally and cut into ∼1-cm pieces. The cut pieces were washed two times at 37°C with 30 ml complete HBSS for 5 min each on a magnetic stir plate (600 rpm), followed by incubation with 50 ml 1-mM DTT for 15 min at 37°C with rotation. After DTT treatment, the tissue was then incubated twice with 30 ml 1-mM EDTA at 37°C with rotation. After each incubation, the epithelial cell layer containing the intraepithelial lymphocytes was removed by aspiration. After the last EDTA incubation, the pieces were washed three times in complete HBSS and placed in 30-ml digestion solution (complete HBSS containing Type III Collagenase and DNase I; Worthington). Digestion was performed by incubating the pieces at 37°C for 1--1.5 h with rotation. The digestion product was collected, filtered through a 70-µM cell strainer (BD), and pelleted by centrifugation. The digestion pellet was then resuspended in 4 ml of 40% Percoll (GE Healthcare) fraction and overlaid on 4 ml of 75% Percoll fraction in a 15-ml conical tube. Percoll gradient separation was performed by centrifugation for 20 min at 2,500 rpm at room temperature. LP cells were collected at the interphase of the Percoll gradient, washed once, and resuspended in either FACS buffer or T cell medium. The cells were used immediately for in vitro stimulation, flow cytometric analysis, or cell sorting. Cells purified by flow cytometry from the LP or total LP cells were cultured at the adjusted density of 10^6^ cells per ml with soluble anti-CD3 (1 µg/ml) overnight, and then cytokines in supernatants (IL-22, IL-17A, and IFN-γ) were analyzed by ELISA (R&D Systems). Intracellular cytokine analysis of total LP cells was performed in cells stimulated in vitro with 50 ng/ml phorbol myristate acetate (Sigma-Aldrich) and 5 µM ionomycin in the presence of Brefeldin A for 6 h. Surface staining was performed with a corresponding cocktail of fluorescently labeled antibodies (eBioscience) for 30 min on ice; followed by permeabilization with Cytofix/Cytoperm solution (BD), and intracellular cytokine staining was performed with fluorescently labeled anti--IL-17 antibody (eBioscience). Immunoblotting for IL-1β was performed using rabbit anti--mouse IL-1β antibody (R&D Systems).

### Real-time PCR analysis.

RNA samples for transcript analysis were isolated from the small intestine or FACS-sorted cells using Total RNA kit I (OMEGA Bio-Tek). Complementary DNAs (cDNAs) were synthesized from and prepared with High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Values were then normalized by the amount of GAPDH in each sample.

### Statistical analyses.

Statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software Inc.). Differences between two groups were evaluated using a Student's *t* test. Differences at P \< 0.05 were considered significant.

The authors thank the University of Michigan Germ-Free Animal Facility and Flow Cytometry Core and the University of Michigan Comprehensive Cancer Center Immunology Core for support; S. Akira and Y. Iwakura for generous supply of mutant mice; S. Koonse for animal husbandry; and G. Chen for review of the manuscript.

This work was supported by grants from the National Institutes of Health (NIH; G. Nuñez). M.H. Shaw was supported by Lung Immunopathology Training grant T32-HL007517 from the NIH. N. Kamada was supported by the Uehara Memorial Foundation and the Crohn's and Colitis Foundation of America. Y.-G. Kim was supported by training funds from the University of Michigan Comprehensive Cancer Center.

The authors declare no competing financial interests.

Author contributions: M.H. Shaw and G. Nuñez designed the research. M.H. Shaw conducted most of the experiments and analyzed data. N. Kamada performed experiments shown in [Fig. 1](#fig1){ref-type="fig"} (G and I). N. Kamada and Y.-G. Kim performed experiments provided as "unpublished data." M.H. Shaw and G. Nuñez wrote the manuscript.

Abbreviations used:EAEexperimental autoimmune encephalomyelitisLPlamina propriaMyD88myeloid differentiation factor 88Rorγtretinoic acid receptor--related orphan receptor γtSFBsegmented filamentous bacteriaSPFspecific pathogen--freesT~H~17 cellssteady-state T~H~17 cellsTLRtoll-like receptor