STAT3 deletion during hematopoiesis causes Crohn’s disease-like pathogenesis and lethality: A critical role of STAT3 in innate

Thomas Welte*, Samuel S. M. Zhang*, Tian Wang†, Zhiyuan Zhang‡, David G. T. Hesslein§, Zhinan Yin†, Arihiro Kano*, Yoshiki Iwamoto*¶,EnLiሻ, Joseph E. Craft†‡, Alfred L. M. Bothwell‡, Erol Fikrig†, Pandelakis A. Koni‡**, Richard A. Flavell‡††‡‡, and Xin-Yuan Fu*‡‡

*Department of Pathology, ‡Section of Immunobiology, ††Howard Hughes Medical Institute, §Department of Cell Biology, †Section of Rheumatology, Department of Medicine, Yale University School of Medicine, New Haven, CT 06520; and ¶Department of Medicine, ʈCardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129

Contributed by Richard A. Flavell, November 22, 2002 Signal transducer and activator of transcription 3 (STAT3) is a key specific abnormalities of myeloid cells have decisive functions in transcriptional mediator for many and is essential for the development of Crohn’s disease. normal embryonic development. We have generated a unique Signal transducers and activators of transcription (STATs) are strain of mice with tissue-specific disruption of STAT3 in bone not only mediators of signaling during immune responses, but marrow cells during hematopoiesis. This specific STAT3 deletion also have roles in development and cell differentiation (12–16). causes death of these mice within 4–6 weeks after birth with They reside in the cytoplasm and become tyrosine-phosphory- Crohn’s disease-like pathogenesis in both the small and large lated by Janus kinases and other protein tyrosine kinases (17, intestine, including segmental inflammatory cell infiltration, ulcer- 18). Tyrosine phosphorylation of STATs leads to their dimer- ation, bowel wall thickening, and granuloma formation. Deletion ization, nuclear translocation, and transcriptional activation of STAT3 causes significantly increased cell autonomous prolifer- (19–22). It is well established that STATs are involved in ation of cells of the myeloid lineage, both in vivo and in vitro. Most multiple steps in adaptive immune responses (23, 24). It will be importantly, Stat3 deletion during hematopoiesis causes overly interesting to investigate functions of STATs in controlling the pseudoactivated innate immune responses. Although inflamma- innate . tory cytokines, including tumor necrosis factor ␣ and IFN-␥, are overly produced in these mice, the NAPDH oxidase activity, In this article, we present evidence that STAT3, which is a which is involved in antimicrobial and innate immune responses, transcriptional mediator for the IL-6 family cytokines, and many is inhibited. The signaling responses to are others such as IL-10, epidermal growth factor, and – changed in the absence of STAT3, leading to enhanced NF-␬B colony-stimulating factor (CSF) (25–28) may have an essential activation. Our results suggest a model in which STAT3 has critical regulatory function in the innate . In particular, roles in the development and regulation of innate immunity, and STAT3 may play a critical role in the control of mucosal immune deletion of STAT3 during hematopoiesis results in abnormalities in tolerance. We generated a unique strain of mice with tissue- myeloid cells and causes Crohn’s disease-like pathogenesis. specific deletion of STAT3 during hematopoiesis. We found that these mice had phenotypes of dramatic expansion of myeloid he innate immune system initiates immediate host responses lineages, causing massive infiltration of the intestine with neu- Tto microbial and acts as an effector by stimulating trophils, , and closely resembling adaptive immune responses (1). The mucosal immune system in Crohn’s disease pathology. This Crohn’s disease-like pathogen- the digestive tract is among the first lines of defense against esis is probably caused by a pseudoactivated innate immune microbial pathogens, which involve both innate and adaptive response to LPS as a result of the STAT3 deletion during immune responses (2). The digestive tract is not normally hematopoiesis. We propose a model that STAT3 mediates affected by despite its exposure to many diverse mucosal immune tolerance during the innate immune response antigens, indicating that there must be an essential mechanism to microbial antigens. that can suppress inflammation by enabling the mucosal immune system to tolerate foreign antigens. One important question is Methods how the mucosal immune system in the gastrointestinal tract Colony Formation Assay. cells were seeded in 1% responds to a variety of antigens and how the decision is made methyl-cellulose in Iscove’s modified Dulbecco’s medium sup- between tolerance and clearance. Disruption of this plemented with 15% FBS, 1% BSA, 10 ␮g͞ml bovine pancreatic balance can lead to disorders in the mucosal immune system that insulin, 200 ␮g͞ml human transferrin, 2 mM L-glutamine, 0.1 cause severe human diseases (3). mM 2-mercaptoethanol, 50 ng͞ml recombinant mouse stem cell For example, in Crohn’s disease and several other inflamma- factor, 10 ng͞ml recombinant mouse IL-3, and 10 ng͞ml recom- tory bowel diseases (IBDs), chronic inflammation can occur in binant human IL-6. parts of the gastrointestinal tract. Regarding molecular etiopa- thology of Crohn’s disease and other IBDs, abnormalities in both innate and adaptive immune responses have been suggested (4, Abbreviations: IBD, inflammatory bowel disease; LPS, lipopolysaccharide; STAT, signal 5). It is likely that the loss of tolerance and uncontrolled transducer and activator of transcription; CSF, colony-stimulating factor; FACS, fluorescence-activated cell sorting; TLR, Toll-like receptor. responses to microbial antigens in the mucosal immune system ¶Present address: Division of Urology and Human and Molecular Genetics Center, Medical is a major cause of Crohn’s disease and other IBDs (6). Recently, College of Wisconsin, Milwaukee, WI 53226-0509. several groups have shown that the NOD2 gene on chromosome **Present addresses: Medical College of Georgia, 1120 15th Street, Augusta, GA 30912 or 16 is implicated in Crohn’s disease (7–9). NOD2 is a member of RIKEN Research Center for and , 1-7-22, Suehiro-cho, Tsurumi-ku, a gene family that mediates response to bacterial lipopolysac- Yokohama, Kanagawa 230-0045, Japan. charide (LPS) (10). Even more interesting, the NOD2 gene is ‡‡To whom correspondence should be addressed. E-mail: richard.fl[email protected] or IMMUNOLOGY expressed in , but not in (11), suggesting [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237137100 PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 1879–1884 Downloaded by guest on October 1, 2021 Bone Marrow Transfer. Recipients (B6) were lethally irradiated (1,100 rad) and received 200,000 donor bone marrow cells through tail vein injection. Eight weeks later bone marrow reconstitution was analyzed by a genomic PCR that detects a donor-derived allele (F allele) with the primers described above.

In Vitro Cultures of Macrophages. Single cell suspensions of were cultured in ␣MEM and 20 ng͞ml CSF-1 as described (30).

Cytometric Bead Array. To measure levels, serum samples (50 ␮l) were added to capture beads (BD Bioscience, San Diego) and mixed with cytokine (anti-IFN-␥, anti-IL-4, anti- tumor necrosis factor ␣, and anti-IL-5); phycoerythrin detection reagent was added to the tube. After a 2-h incubation samples were analyzed with fluorescence-activated cell sorting (FACS). Absolute values were obtained by comparisons with standards.

Statistical Analysis. P values were calculated with a nonpaired Student’s t test.

Additional Methods. For descriptions of other methods used see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org. Results A Mouse Strain with a Conditional STAT3 Disruption Developed Gradual Lethality After Birth. We have generated a STAT3 allele in which exons 18–20 are flanked by loxP sequences (STAT3-F, Fig. 6, which is published as supporting information on the PNAS web site). Removal of exons 18–20, which encode the Src homology 2 domain of STAT3, is expected to eliminate the function of the protein. To study immunological functions of STAT3, we chose here a mouse strain for tissue-specific gene deletion where Cre expression is driven by a TIE2 gene promoter͞enhancer cassette (Tie2-Cre, Fig. 6). The TIE2 gene promoter drives Cre expression in bone marrow and endothelial cells (32). In two steps of breeding STAT3-loxP with Tie2-Cre, we obtained mice that are homozygous for STAT3-loxP (F allele) and Tie2-Creϩ (C allele), which are conditional STAT3 knock- out mice and designated as STAT3-CFF. These mice were obtained at the expected Mendelian ratio and appeared normal at birth, indicating the absence of severe embryonic deficiencies of STAT3-CFF mice. However, at 3–4 weeks of age, offspring with the STAT3 deletion were smaller with reduced body weight compared with their siblings. All STAT3-CFF animals appeared fragile and weak by 4–6 weeks after birth. Of eight animals tested, none survived Ͼ8 weeks, whereas WT (STAT3-C, or Fig. 1. STAT3 deficiency during hematopoiesis results in Crohn’s disease-like STAT3-FF) or heterozygous (STAT3-CF͞ϩ) littermates were pathology. (a–j) Ulcers (arrow 1), transmural infiltration (arrow 2), and gran- viable and developed normally. uloma-like structures (arrow 3) in the ileocecal area of STAT3-CFF mice in c, d, and f–j. For comparison, the normal phenotype is depicted in a, b, and e.(c and d) Ileum of STAT3-CFF mouse. (e) Cecum of a control littermate and (f)ofa Crohn’s Disease-Like Pathogenesis in STAT3-CFF Mice. To determine STAT3-CFF mouse. (Magnifications: ϫ40, a–d; ϫ10, e and f.) (g–j) Higher- the possible cause of death, necropsies were performed. Obser- magnification views (ϫ100, hematoxylin and eosin). (g) Crypt abscesses with vation of the peritoneum revealed that the ileum was fused to the necrotic and monocytes (arrow 4), (h) high frequency of mitosis in peritoneal wall in severely sick STAT3-CFF animals. This find- the epithelium (arrow 5), (i) a marked infiltration of neutrophils, macro- ing is an indication of a previous perforation or transmural phages, and eosinophils (arrow 6) and few lymphocytes, edema of the lamina inflammation of the gut. Histological sections of the ileum of propria, (j) epithelioid cells with eosinophilic cytoplasm (arrow 7) and other control (Fig. 1 a and b) and STAT3-CFF animals (Fig. 1 c and inflammatory cells in the lamina propria can be identified. (k) Intestinal d) indicated that the ileum was dramatically affected by STAT3 mesentry of control and (l) STAT3-deleted mice. Infiltration of neutrophils, deletion. Significantly, changes were present in all four major macrophages, eosinophils (arrow 8 in l), and other cell types in the mesenteric fat tissue of STAT3-CFF mouse (ϫ40, hematoxylin and eosin). Six sex-matched layers: mucosa, submucosa, smooth muscle, and serosa, ulcer- pairs of control (STAT3-FF) and STAT3-CFF littermates between 4 and 6 weeks ation (Fig. 1c, arrow 1), and loss of mucosal texture and of age were analyzed with similar results. IBD also spreads to the colon (n). transmural inflammation (Fig. 1c, arrow 2). Higher-magnifica- Note bowel wall thickening with mucosal erosion (arrow 9) and transmural tion views revealed striking neutrophilic and monocytic infiltra- inflammation (arrow 10) in STAT3-CFF mice (ϫ40, hematoxylin and eosin). tion in the submucosal, muscular and serosa layers (Fig. 1d). Granuloma-like structures were also observed. The histology of the cecum area showed similar changes. (compare Fig. 1 e and f). Many further typical signs for IBD were Ulceration and a transmural inflammation of all layers occurred. found in this area, such as crypt abscesses with necrotic neutro- The total thickness of the bowel wall was markedly increased phils and monocytes (arrow 4 in Fig. 1 g and h) and marked

1880 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237137100 Welte et al. Downloaded by guest on October 1, 2021 ZEGϩTie2-Creϩ pups were isolated and classified with cell type and stage-specific antibodies. Then we examined these cells for GFP expression by FACS analysis. We found that maturation marker-positive cells (Mac1ϩ for myeloid) show high GFP expression. More than 80% of cells expressed GFP in each case (Fig. 7, which is published as supporting information on the PNAS web site, and results not shown). Different precursor cell populations, such as c-kitϩ lineage marker (B220, CD4, CD8, TER119, Mac-1)Ϫ cells, ScaIϩ lineage marker (B220, CD4, CD8, TER119, Mac-1)Ϫ, and B220ϪCD43ϩ cells also expressed GFP (Ͼ75%, Ͼ60%, and Ͼ75% of the cells, respectively, Fig. 7 and results not shown). Similarly, high activity of Tie2Cre was detected in CD144ϩPECAM-1ϩ in the (endothelial cells, data not shown). Therefore this Tie2Cre strain mediates gene deletion of loxP-containing alleles during hematopoietic devel- opment starting at early precursor stages. As mentioned above, we expect that Cre expression should cause deletion of floxed STAT3 and the genotype (CFF) leads to conditional STAT3 gene ablation (Fig. 6). To observe whether gene deletion occurs early in the animal’s life, spleens of 2-day-old pups Fig. 2. The inflammatory phenotype is not restricted to the gut but also were analyzed. Cells were fractioned, and the cell pellet containing occurs in liver. (a–c) Tissue sections of liver, kidney, and heart of control red blood cells and the interphase containing white blood cells were littermates. (Magnifications: ϫ40.) (d–f) STAT3-CFF littermates. (d) The liver of compared (Fig. 7). A blot with STAT3 demonstrated only STAT3-CFF mice was infiltrated around the portal vein by neutrophils, mono- minor deletion in the fraction enriched for red blood cells and cytes, and other cell types (arrow 1); hepatocytes were of normal appearance efficient deletion of STAT3 in the fraction enriched for white blood (arrow 2) (ϫ40, hematoxylin and eosin). (e) Kidney had normal glomeruli cells (Fig. 7) at this age. Furthermore, specific cell types from the ϫ (arrow 3) and tubular structures without inflammatory cell infiltration ( 40, bone marrow of 3-week-old animals were analyzed to address hematoxylin and eosin). (f) Heart showed normal myocardial fibers (arrow 4) without inflammatory cell infiltration (ϫ40, hematoxylin and eosin). Five age- whether STAT3 gene deletion occurred in bone marrow cells of different lineages and at different stages of development. Different and sex-matched pairs were analyzed. Pathologic alteration of the liver was ϩ observed in all STAT3-CFF animals examined. cell populations were isolated by FACS sorting. Sorted Mac1 (myeloid cells) and c-kitϩ lineage marker precursor cells were analyzed by Western blotting with an anti-STAT3 antibody. Ex- infiltration of neutrophils, macrophages and eosinophils (arrow pression of STAT3 was found in myeloid and precursor cells in 2 in Fig. 1i). Epitheloid cells with an eosinophilic cytoplasm were control littermates (STAT3-FF) (Fig. 7) and was deleted by Tie2- aggregated and reminiscent of giant fused cells that have been Cre to undetectable levels in these cell types in STAT3-CFF mice observed in Crohn’s disease (arrow 7 in Fig. 1j). Having estab- (Fig. 7). lished the severe leukocyte infiltration of the intestine, we These results were confirmed by performing a genomic PCR examined the possible route of entry of inflammatory cells and analysis with DNA isolated from sorted cell populations (Fig. 6). investigated the intestinal mesentry (Fig. 1 k and l). The fat tissue of the mesentry was massively invaded with neutrophils, mac- STAT3 Deletion Causes Abnormal Development of Myeloid Cells in rophages, eosinophils (arrow 8 in Fig. 1l), and other cell types STAT3-CFF Mice. The striking immune response phenotype ob- (compare with control, Fig. 1k, and STAT3-CFF, Fig. 1l). served in STAT3-CFF mice could be caused by a role of STAT3 Another area of the gastrointestinal tract that is susceptible to in the development of the innate immune system. In particular, IBD is the colon. We demonstrate that this area of the gut is also STAT3 may control myeloid cell proliferation and differentia- affected by pathological phenotypes in a representative 4-week- tion and sensitivities to microbial antigens. old STAT3-CFF mouse. In comparison to the control (Fig. 1m), To examine these possibilities we first determined the cellular composition in the bone marrow by FACS analyses using a panel the colon of STAT3-CFF animals (Fig. 1n) showed bowel wall of antibodies against cell type and stage-specific surface anti- thickening, mucosal erosion, transmural inflammation affecting ϩ ϩ gens. GR-1 Mac1 cells represent the lineage. We all layers, edema in the submucosa, and serosa thickening. observed an expansion of this cell type typically from 20% in We have also examined other organs (liver, kidney, and heart) controls and heterozygous (STAT3-CFϩ) animals to 35% in for pathological features (Fig. 2). In the liver, infiltration of STAT3-CFF homozygous knockout bone marrow in all mice was observed around the portal vein (arrow 1 in (n ϭ 15) analyzed at 3–6 weeks of age (Fig. 3a). A similarly Fig. 2d). Similar to the gastrointestinal tract, neutrophils and skewed cellular distribution is observed at an age when there are monocytes could be distinguished in the infiltrate. Heart and no outward signs of disease (3 weeks) or at later stages (4–6 kidney appeared normal in these animals (Fig. 2 e and f). weeks). By comparison, the relative abundance of TER119ϩ cells (erythroid) is not changed in bone marrow from these mice (Fig. Specific STAT3 Deletion in Myeloid Cells in Mice with Homozygous F 3a). These experiments revealed an abnormal expansion of a Allele and Tie2-Cre (STAT3-CFF). As shown above, the STAT3 dele- myeloid lineage in the bone marrow in the absence of STAT3. tion mediated by Tie2-Cre in STAT3-CFF mice leads to severe Colony formation assays revealed that the number of prolifer- Crohn’s disease-like phenotypes. Interestingly, myeloid cells, rather ating precursors was slightly increased in the bone marrow (Fig. than lymphocytes, are the major cell populations in the inflamma- 3b), indicating that STAT3 may have a minor effect on control tory infiltrates of the gastrointestinal tract. This observation indi- of the number of progenitors generated in the bone marrow, but cates that STAT3 deletion causes abnormalities in myeloid cells. a major effect on expansion and differentiation at a later stage. To determine cell and stage specificity of the Tie2-Cre used to delete STAT3 in this study, we analyzed a reporter mouse. The Cell Autonomous Overproduction of Myeloid Cells with STAT3 Dele- ZEG reporter mouse, which expresses GFP as a measure of Cre tion. We next asked whether myeloid overproliferation is a IMMUNOLOGY activity, was crossed with Tie2-Cre mice. Bone marrow cells of cell-autonomous function. Bone marrow of STAT3-deficient

Welte et al. PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 1881 Downloaded by guest on October 1, 2021 Fig. 4. STAT3 regulates the generation of the myeloid lineage in a cell autonomous fashion in vivo and in vitro.(a) Bone marrow (BM) was trans- ferred from a donor with control genotype (Center, STAT3-FF) or from a STAT3-CFF donor (Right) into lethally irradiated recipients. The presence of donor-derived bone marrow was determined after 8 weeks. Bone marrow was analyzed for the presence of GR-1ϩMac1ϩ cells and compared with bone marrow of an age-matched untreated animal (Left). Experiments with three controls and three STAT3-CFF animals showed an increased percentage of GR-1ϩMac1ϩ cells in the absence of STAT3. (b) Bone marrow from control (plates 1 and 3, STAT3-FF) and STAT3-deleted (plates 2 and 4, STAT3-CFF) littermates was cultured for 1 and 3 days in the presence of CSF-1 (30 ng͞ml). Light microscopic pictures were taken showing a dramatic increase in the generation of attached cells in cultures of STAT3-deficient bone marrow. FACS with Mac1 antibody confirmed that these cells were macrophages. Three repeats showed similar results. (Magnification: ϫ2.) Fig. 3. In the absence of STAT3, hematopoietic development is skewed toward the myeloid lineage at an early stage. (a) The presence of the myeloid ϩ ϩ ϩ stimulate the expansion of macrophages. At early time points (Fig. lineage (GR-1 Mac1 ) and erythroid lineage (TER119 ) in the bone marrow of ϩ sex-matched littermates was analyzed by FACS staining. The genotype of the 4b, plates 1 and 2) the number of attached Mac1 cells was low control is STAT3-FF. Heterozygous is STAT3-CFϩ and knockout (KO) is STAT3- regardless of the genotype. Stimulation with CSF-1 for 3 days led ϩ CFF. A result with typical ratios of GR-1ϩMac1ϩ and TER119ϩ cells is shown. to an increase of attached Mac1 cells, indicating that macrophages Three repeats with heterozygous animals and at least 15 repeats with control were generated in the cultures. When STAT3-deficient splenocytes and STAT3-CFF mice gave similar ratios between the individual cell lineages. were cultured, a much higher number of attached Mac1ϩ cells were (b) Bone marrow (BM) was cultured for 7 days in methyl-cellulose in the present compared with controls (Fig. 4b, compare plates 3 and 4). presence of cytokines (IL-3, IL-6, stem cell factor, and insulin). The numbers of colonies formed were counted. Results obtained with four sex-matched pairs Thus, the propensity of generating macrophages in response to of control (STAT3-FF) and STAT3-CFF littermates are shown. The difference in CSF-1 is negatively regulated by STAT3. the colony numbers of control and STAT3-CFF mice was not statistically significant. (c) FACS analyses with antibodies against c-Kit and different The STAT3 Gene Deletion Causes Dysfunctional Innate Immunity. To lineage markers (Mac1, B220, CD4, CD8, and TER119). The percentage of c-Kitϩ elucidate the mechanisms that could be involved in the gener- lineage markerϪ cells in the bone marrow of STAT3-CFF mice was evaluated ation and propagation of Crohn’s disease in the gut and activa- and compared with sex-matched littermates. The results of five independent tion of the innate immune system, we asked how Toll-like experiments are shown. receptor (TLR) signaling in STAT3-CFF mice would be regu- lated. TLRs have been recognized as an important family of receptors for the activation of innate immune cells in response mice was transferred to lethally irradiated WT mice. The bone to bacteria and other pathogens (33). LPS, for example activates marrow transfer rescued the survival of the animals, indicating TLR4. Phosphorylation of I␬B, degradation of I␬B, and conse- a successful procedure. Eight weeks after transfer, the bone quent activation of NF-␬B are downstream events of TLR marrow of the recipients was reconstituted and was derived stimulation. Activation and differentiation of dendritic cells by mainly from transferred cells. A genomic PCR control showed TLR signaling is an initial event of innate immune responses. that the majority of bone marrow cells had the genotype of the LPS treatment of bone marrow-derived dendritic cells induced donor (results not shown). Mice reconstituted with STAT3- I␬B phosphorylation and its degradation (Fig. 5a, compare lanes deficient bone marrow (STAT3-CFF) showed an increased ␬ ϩ 1–3). In comparison to control, I B phosphorylation normalized proportion of the myeloid lineage (Mac1 ) in bone marrow and to I␬B protein level was induced 3-fold more in STAT3-CFF spleen (1.6- and 2.5-fold increase, respectively) that was very mice after a 1-h stimulation (Fig. 5a, lanes 4–6). Gel-shift similar to what was seen in the original STAT3-deficient mice. experiments with an NF-␬B DNA binding site showed that the As a control, transfer of control bone marrow (STAT3-FF) DNA binding activity of NF-␬B family members was enhanced ϩ yielded ratios of Mac1 cells after 8 weeks as found in untreated in the absence of STAT3 after stimulation (Fig. 8, which is mice (Fig. 4a). published as supporting information on the PNAS web site). A We confirmed the cell autonomous proliferation of myeloid cells similar result was obtained on stimulating splenic B cells (Fig. 8). in in vitro studies of their growth. Spleen cells from STAT3-CFF Previous studies have shown that the generation of reactive mice and control mice were cultured in the presence of CSF-1 to oxygen species by NADPH oxidase is an important response

1882 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237137100 Welte et al. Downloaded by guest on October 1, 2021 strains with a conditional STAT3 gene knockout, our strain has a rather complete deletion of STAT3 in the bone marrow cells, which causes novel and severe phenotypes, including a widespread in- flammatory disease in the digestive tract and develops close to 100% lethality at an early age (4–6 weeks after birth). The pathological abnormalities resemble some of the hall- marks for Crohn’s disease (38), making these mice a valuable model to understand the mechanisms underlying Crohn’s dis- ease and related intestinal bowel diseases. Presumably mucosal interstitia of the gastrointestinal tract are continuously exposed to a variety of foreign antigens. However, no severe inflamma- tory response against these foreign antigens occurs, indicating that the mucosal immune system in the gastrointestinal tract has developed unique mechanisms for immune tolerance. When inflammatory responses occur during invasion by a pathogen, the same mechanism may also control the immune response to a limited location and time and enable the system to turn off the response when pathogens are cleared and to suppress the hypersensitive innate immune responses to regular microflora in the gastrointestinal tract. Crohn’s disease and other IBDs likely result from dysfunction of this ‘‘turn-off’’ mechanism. Currently little is known about the molecular mechanisms by which mucosal immune tolerance is regulated. Our model is that STAT3 is a key regulator of innate immune responses. We identified at least three stages that involve STAT3 in orchestrating tolerance in the mucosal immune system of the gut. First, dendritic cells and B cells that lack STAT3 show an enhanced immediate response to bacterial LPS by generating higher NF-␬B activities after stimulation. Because these cell types are critical in the initial uptake of antigen and trigger further responses of other cell types, the observed increased TLR signaling could explain how the system initially becomes sensitive to the otherwise innocuous microflora in the gastro- intestinal tract. We have found that in STAT3-deficient dendritic cells the LPS-induced I␬B␣ phosphorylation, leading to subse- Fig. 5. STAT3 regulates crucial functional responses of the innate immune quent NF-␬B activation, is Ϸ3-fold higher in STAT3-CFF mice. system. (a)I␬B protein and phosphorylation levels were measured by Western Whereas such a change may be considered to be moderate, it blotting with specific antibodies to I␬B and phospho-I␬B before and after 10-min ͞ could be of striking significance in a system such as the gut, and 1-h LPS (10 ng ml) stimulation of bone marrow-derived myeloid cells cul- where TLR stimulation could occur chronically and repeatedly. tured in granulocyte͞macrophage-CSF as indicated (FF, control mice; CFF, STAT3- ␬ CFF mice). (b) NADPH oxidase activity was measured in neutrophils isolated from The down-regulation of LPS-induced NF- B activation by whole blood samples of 4- to 5-week-old mice. Five controls (shaded bar) were STAT3 could be one important factor in the maintenance of compared with five sex-matched littermates (hatched bars). The difference be- tolerance and could be of general relevance for Crohn’s disease. tween control and STAT3 knockout mice was statistically significant (five pairs This notion is supported by the recent discovery that NOD2 has analyzed). P value of Student’s t test was P Ͻ 0.004. (c) Sera from 4- to 5-week-old been identified as the first susceptibility gene for Crohn’s disease mice were collected. IFN-␥, IL-4, tumor necrosis factor ␣ (TNF-␣), and IL-5 levels (7–9). NOD2 was shown to also regulate NF-␬B activation. were measured. The results with three matched pairs of control (shaded bars) and Second, deletion of STAT3 causes overproliferation of mac- STAT3-CFF littermates (open bars) are shown. rophages and neutrophils, which could be the second factor responsible for an enhanced response of the innate immune induced by the innate immune system. NADPH oxidase activity system. This event is triggered by the cytokines -CSF is increased in normal mice during an inflammatory process and granulocyte-CSF, respectively. Our finding that STAT3 (34–36). We measured NADPH oxidase activity of neutrophils negatively regulates proliferation in response to these cytokines isolated from whole blood samples of WT control mice by using is consistent with a recent report of Lee et al. (39), in which a Fc-Oxyburst analysis (37). In this assay, NADPH oxidase activity negative role of STAT3 in the growth response to granulocyte- is Ͼ90% positive. By contrast, in the STAT3-deficient mice CSF is suggested. However, the study by Lee et al. did not expand NADPH oxidase activity was reduced to 45% positive (Fig. 5b). into IBDs. We show here that myeloid cells are recruited in high This finding indicates that STAT3-CFF mice have decreased numbers to inflammatory sites in STAT3-CFF mice. The im- host defense ability. As a consequence, it is possible that these portance of the inhibition of myelopoiesis by STAT3 lies in the STAT3-deficient mice are prone to further up-regulate their fact that it could moderate and prevent overgeneration of these response to an insult. It is expected that the concentrations of cells under nonpathological conditions and during clearance of inflammatory cytokines will increase during this response. We tested several cytokines in the serum of the mice. Tumor necrosis the pathogen. Furthermore, our analysis and our unpublished factor ␣ and IFN-␥ were present at increased levels in the results show that STAT3 inhibits not only the generation of STAT3-deficient mice (Fig. 5c), indicating an ongoing deregu- neutrophils but also other types of myeloid cells (monocytes, lated T helper 1-type immune response. myeloid dendritic cells, and osteoclasts). These findings chal- lenge the current paradigm that positions STAT3 as an oncogene Discussion for promoting cell proliferation. In contrast, we propose here In this study we generated mice lacking STAT3 in the hematopoi- that STAT3 is a negative regulator of cell growth under physi- IMMUNOLOGY etic system to analyze functions of STAT3 in vivo. Unlike previous ological conditions.

Welte et al. PNAS ͉ February 18, 2003 ͉ vol. 100 ͉ no. 4 ͉ 1883 Downloaded by guest on October 1, 2021 Third, we found that STAT3 regulates critical effector functions sponses in Crohn’s disease and related animal models. Our studies of the activated innate immune system. This finding is documented have indicated that activated myeloid cells have a more prominent by two examples. First, deletion of STAT3 causes compromised role than lymphocytes in the initiation of the disease. There are few NADPH oxidase activities in neutrophils (Fig. 5b), thereby ham- lymphocytes present in the infiltrated areas, yet the inflammatory pering the efficacy of the innate immune system to battle infectious responses are severe and sufficient for generation of the disease microbes. Difficulties in clearance of microbial antigens will further phenotypes. This observation is supported by the studies on NOD2, induce stronger and continuous responses, resulting in escalating the susceptibility gene for Crohn’s disease. Interestingly, NOD2 is inflammatory reactions. Second, the literature on mice whose expressed only in monocytes but not in lymphocytes, consistent with NADPH oxidase subunits were gene targeted is in line with our the essential roles of myeloid cells in the disease. Furthermore we reasoning, because those mice develop a chronic inflammatory analyzed responses of STAT3-CFF mice in a tumor challenge syndrome similar to chronic granulomatous disease in the absence experiment and found no evidence for an increased proliferation of of functional NADPH oxidase (40). T cells cultured ex vivo from draining lymph nodes (data not Takeda et al. (41) provided an earlier example of effector shown). This result may indicate that T cell responses per se are not functions regulated by STAT3 in myeloid cells that causes a mild enhanced in the absence of STAT3, consistent with previous IBD (29). This IBD was attributed to defects in the function of reports on STAT3-deficient T cells (31). macrophages in response to IL-10. In contrast, we presented data Our major conclusion is that STAT3 regulates innate immune here suggesting that STAT3 deletion causes additional abnormal- responses at several levels, through down-regulating NF-␬B ities in the regulation of innate immune responses. The changes in activation by TLRs, through inhibiting the growth response to LPS signaling, macrophage-CSF signaling, and NADPH oxidase cytokines such as granulocyte-CSF and macrophage-CSF, and function all could significantly contribute to the observed inflam- through regulating an effector function of neutrophils, the matory phenotype. Although the enhanced phenotype of the current STAT3-CFF mice could be caused by differences in strain NADPH oxidase activity. This finding suggests that several backgrounds and efficiencies in gene deletion, it is more likely pathways important for the tolerance of the innate immune caused by the fact that in the current model STAT3 was not only system are coordinately regulated by STAT3 and may explain the deleted in macrophages and neutrophils, but also other cell types severity of the Crohn’s-like inflammatory disease observed in that develop innate immune responses and respond to microbial STAT3-deficient mouse strain in the present study. challenge (endothelial cells, dendritic cells, and B cells). By using an We thank Drs. Joe Madri and Jordan Porber for discussions, Drs. A. IL-10-neutralizing antibody we obtained evidence for an IL-10- ͞ independent function of STAT3 in the LPS response of B cells Nagy and C. Lobe for Z EG mice, Marie Robert for help with diagnosis, and Qian Gao and Michael Wolfgang for reading the manuscript and (results not shown). Therefore we believe that the negative role of providing comments. T. Welte is an Austrian Programme for Advanced STAT3 for innate immunity is widespread and not restricted to the Research and Technology Fellow of the Austrian Academy of Sciences previously proposed mechanism that operates specifically in mac- (Osterreichische Akademie Der Wissenschaften). R.A.F. is an Investi- rophages and down-regulates cytokine production through IL-10 gator of the Howard Hughes Medical Institute. X.-Y.F. was a recipient during the later stage of . of a Career Development Award from the National Institutes of Health. Our results further contrast the view that activated CD4 T This work was supported by National Institutes of Health Grants lymphocytes are the major players during the inflammatory re- AI34522 and AR44906 (to X.-Y.F.).

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