Assessing an alpha-defensin lateral flow device for diagnosing septic arthritis: reporting on a false-negative case and a false-positive case.

Alpha-defensin (αD), an antimicrobial peptide released by neutrophils in response to bacterial pathogens, was proposed as a novel diagnostic biomarker in synovial fluid. Several reports have shown that αD can serve as a reliable biomarker in the diagnosis of periprosthetic joint infection (PJI). We assessed whether αD could also serve to diagnosis of septic arthritis, a similarly difficult to diagnose PJI. To our knowledge, besides PJI, few reports exist assessing the utility of αD for septic arthritis. We have attempted to diagnose several cases of suspected septic arthritis using the Synovasure® αD detection lateral flow device. We report a false-positive case and a false-negative case. The false-negative case we experienced was caused by Staphylococcus capitis, which is coagulase-negative, and possibly represents a low virulence micro-organism infection. The false-positive case was ultimately diagnosed as seronegative rheumatoid arthritis and possessed calcium pyrophosphate depositions. False positives have been suggested to occur in conditions where neutrophils are mobilised. As for PJI, in cases where diagnosis is difficult, αD can be an additional diagnostic indicator. However, making a definitive diagnosis using the αD lateral flow device alone was found to be difficult. The utility of αD in assessing septic arthritis is inconclusive; therefore, larger prospective clinical studies should be considered for a better assessment.

Gene Signatures of Early Response to Anti-TNF Drugs in Pediatric Inflammatory Bowel Disease.

Around a 20-30% of inflammatory bowel disease (IBD) patients are diagnosed before they are 18 years old. Anti-TNF drugs can induce and maintain remission in IBD, however, up to 30% of patients do not respond. The aim of the work was to identify markers that would predict an early response to anti-TNF drugs in pediatric patients with IBD. The study population included 43 patients aged <18 years with IBD who started treatment with infliximab or adalimumab. Patients were classified into primary responders (n = 27) and non-responders to anti-TNF therapy (n = 6). Response to treatment could not be analyzed in 10 patients. Response was defined as a decrease in over 15 points in the disease activity indexes from week 0 to week 10 of infliximab treatment or from week 0 to week 26 of adalimumab treatment. The expression profiles of nine genes in total RNA isolated from the whole-blood of pediatric IBD patients taken before biologic administration and after 2 weeks were analyzed using qPCR and the 2-∆∆Ct method. Before initiation and after 2 weeks of treatment the expression of SMAD7 was decreased in patients who were considered as non-responders (p value < 0.05). Changes in expression were also observed for TLR2 at T0 and T2, although that did not reach the level of statistical significance. In addition, the expression of DEFA5 decreased 1.75-fold during the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders. Expression of the SMAD7 gene is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD. TLR2 and DEFA5 need to be validated in larger studies.

A mutated rabbit defensin NP-1 produced by Chlorella ellipsoidea can improve the growth performance of broiler chickens.

The demand for alternatives to antibiotics to improve the growth performance of food animals is increasing. Defensins constitute the first line of defence against pathogens in the innate immune system of animals and humans. A transgenic Chlorella ellipsoidea strain producing mNP-1 (a mutated rabbit defensin NP-1) was previously obtained in our laboratory. In this study, a process for producing the transgenic strain on a large scale was developed, and the C. ellipsoidea strain producing mNP-1 was used as a feed additive to improve the health and growth performance of chickens. The volume of C. ellipsoidea producing mNP-1 can be scaled up to 10,000 L with approximately 100 g/L dry biomass, and the mNP-1 content of transgenic microalgal powder (TMP) was 90-105 mg/L. A TMP-to-regular feed ratio of 1‰, as the optimal effective dose, can promote the growth of broiler chickens by increasing weight by 9.27-12.95%. mNP-1 can improve duodenum morphology by promoting long and thin villi and affect the microbial community of the duodenum by increasing the diversity and abundance of beneficial microbes. These results suggested that transgenic Chlorella producing mNP-1 can be industrially produced and used as an effective feed additive and an alternative to antibiotics for improving the health and growth performance of broiler chickens or other types of food animals/poultry.

Nutrient Sensing by the Intestinal Epithelium Orchestrates Mucosal Antimicrobial Defense via Translational Control of Hes1.

Metabolic programs and host defense are highly integrated to ensure proper immune responses during stress. Central to these responses, mTOR regulates immune functions by sensing and integrating environmental cues, yet how these systems are coordinated at the intestinal surface remains undefined. We show that the antimicrobial peptide α-defensin is functionally sustained during nutrient deprivation because of regulation of the defensin-processing enzyme MMP7 by microbiota- and host-derived factors. Unlike other antimicrobial peptides, the MMP7-α-defensin axis remains active during nutrient fluctuations, providing essential protection against enteric pathogens. Sustained Mmp7 expression requires the microbiota and is mediated by de-repression of the transcription activator Atoh1 upon attenuation of the transcriptional repressor Hes1 in intestinal epithelial cells. Hes1 levels are regulated via mTOR and controlled translationally, constituting a metabolism-translation-transcription loop. Disrupting this loop by supplying nutrients paradoxically compromises antibacterial defense. Together, these results uncover a regulatory circuit that couples host nutrient status to epithelial antimicrobial immunity.

Enteric α-defensins on the verge of intestinal immune tolerance and inflammation.

The gut is the biggest immune organ in the body that encloses commensal microbiota which aids in food digestion. Paneth cells, positioned at the frontline of host-microbiota interphase, can modulate the composition of microbiota. Paneth cells achieve this via the delivery of microbicidal substances, among which enteric α-defensins play the primary role. If microbiota is dysregulated, it can impact the function of the local mucosal immune system. Importantly, this system is also exposed to an enormous number of antigens which are derived from the gut-resident microbiota and processed food, and may potentially trigger undesirable local inflammatory responses. To understand the intricate regulations and liaisons between Paneth cells, microbiota and the immune system in this intestinal-specific setting, one must consider their mode of interaction in a wider context of regulatory processes which impose immune tolerance not only to self, but also to microbiota and food-derived antigens. These include, but are not limited to, tolerogenic mechanisms of central tolerance in the thymus and peripheral tolerance in the secondary lymphoid organs, and the intestine itself. Defects in these processes can compromise homeostasis in the intestinal mucosal immunity. In this review, which is focused on tolerance to intestinal antigens and its relevance for the pathogenesis of gut immune diseases, we provide an outline of such multilayered immune control mechanisms and highlight functional links that underpin their cooperative nature.

Gamma-Aminobutyric Acid (GABA) Attenuates Ischemia Reperfusion-Induced Alterations in Intestinal Immunity.

The aims of this study were to determine the effects of gamma-aminobutyric acid (GABA) on immunoglobulin A (IgA) secretion from Peyer's patch (PP) cells; to assess rat alpha-defensin-5 (RD-5) expression in the rat small intestine; and to determine the effect of GABA on intestinal ischemia reperfusion (I/R) injury-induced intestinal innate immunity. We found that GABA caused an increase in IgA secretion in the presence and absence of lipopolysaccharide (LPS). Moreover, GABA also significantly increased the mRNA levels of RD-5 and superoxide dismutase (Sod) 1, 3. Intestinal I/R was induced by a 30-min occlusion of the superior mesenteric artery followed by a reperfusion for 60-min. This led to a significant decrease in IgA secretion, and mRNA levels of RD-5 and Sod 1-3 in the ileum. On the other hand, administration of GABA before I/R induction had a significant protective effect against oxidative injury and attenuated the effects on intestinal immunity.

Silk-Based Antimicrobial Polymers as a New Platform to Design Drug-Free Materials to Impede Microbial Infections.

Surgical site infections (SSI) represent a serious health problem that occur after invasive surgery, thus new antimicrobial biomaterials able to prevent SSI are needed. Silks are natural biopolymers with excellent biocompatibility, low immunogenicity and controllable biodegradability. Spider silk-based materials can be bioengineered and functionalized with specific peptides, such as antimicrobial peptides, creating innovative polymers. Herein, we explored new drug-free multifunctional silk films with antimicrobial properties, specifically tailored to hamper microbial infections. Different spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6mer and 15mer, 27 and 41 kDa proteins, respectively) were fused with the two antimicrobial peptides, Hepcidin (Hep) and Human Neutrophil peptide 1 (HNP1). The self-assembly features of the spider silk domains (ß-sheets) were maintained after functionalization. The bioengineered 6mer-HNP1 protein demonstrated inhibitory effects against microbial pathogens. Silk-based films with 6mer-HNP1 and different contents of silk fibroin (SF) significantly reduced bacterial adhesion and biofilm formation, whereas higher bacterial counts were found on the films prepared with 6mer or SF alone. The silk-based films showed no cytotoxic effects on human foreskin fibroblasts. The positive cellular response, together with structural and antimicrobial properties, highlight the potential of these multifunctional silk-based films as new materials for preventing SSI.

Effects of vitamin D status on oral health.

Normal humans of all ages have the innate ability to produce vitamin D following sunlight exposure. Inadequate vitamin D status has shown to be associated with a wide variety of diseases, including oral health disorders. Insufficient sunlight exposure may accelerate some of these diseases, possibly due to impaired vitamin D synthesis. The beneficial effects of vitamin D on oral health are not only limited to the direct effects on the tooth mineralization, but are also exerted through the anti-inflammatory functions and the ability to stimulate the production of anti-microbial peptides. In this article, we will briefly discuss the genesis of various oral diseases due to inadequate vitamin D level in the body and elucidate the potential benefits of safe sunlight exposure for the maintenance of oral and general health.

Amyloid formation: functional friend or fearful foe?

Amyloid formation has been most studied in the context of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, as well as in amyloidosis. However, it is becoming increasingly clear that amyloid is also present in the healthy setting; for example nontoxic amyloid formation is important for melanin synthesis and in innate immunity. Furthermore, bacteria have mechanisms to produce functional amyloid structures with important roles in bacterial physiology and interaction with host cells. Here, we will discuss some novel aspects of fibril-forming proteins in humans and bacteria. First, the amyloid-forming properties of the antimicrobial peptide human defensin 6 (HD6) will be considered. Intriguingly, unlike other antimicrobial peptides, HD6 does not kill bacteria. However, recent data show that HD6 can form amyloid structures at the gut mucosa with strong affinity for bacterial surfaces. These so-called nanonets block bacterial invasion by entangling the bacteria in net-like structures. Next, the role of functional amyloid fibrils in human semen will be discussed. These fibrils were discovered through their property to enhance HIV infection but they may also have other yet unknown functions. Finally, the role of amyloid formation in bacteria will be reviewed. The recent finding that bacteria can make amyloid in a controlled fashion without toxic effects is of particular interest and may have implications for human disease. The role of amyloid in health and disease is beginning to be unravelled, and here, we will review some of the most recent findings in this exciting area.

Morphological and functional adaptations of Fusobacterium nucleatum exposed to human neutrophil Peptide-1.

BACKGROUND AND OBJECTIVE: We recently demonstrated that Fusobacterium nucleatum can resist to human neutrophil peptide (HNP)-1 by decreasing its membrane permeability and increasing its proliferation and biofilm formation. In this continuation study, we aimed to further evaluate and explain these resistance properties by determining the morphological and functional adaptations of F. nucleatum, using transmission electron microscopy (TEM). MATERIALS AND METHODS: Cultures of the type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were incubated without (0 µg/ml) or with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml). Membrane morphology and thickness, and cell (visualized by TEM), planktonic growth (measured in colony forming units), and biofilm formation (measured as total mass) were analyzed. Scrambled HNP-1 was used in planktonic growth and biofilm formation studies as a negative control. RESULTS: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. Intracellular granules became increasingly visible in the strain ATCC 25586 with increasing peptide concentrations. With increased concentrations of HNP-1, planktonic growth of the two clinical strains was significantly enhanced (P < 0.001) and of the type strain significantly suppressed (P < 0.01). HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 (P < 0.01) and 9508 (P < 0.001) significantly. Scrambled HNP-1 showed no effect on planktonic growth or biofilm formation of the tested strains. DISCUSSION: F. nucleatum has the ability to withstand the lethal effects of HNP-1, and the ultrastructural changes on bacterial membrane and cytoplasm may play role in this adaptive process.

Chronic HIV Infection Is Associated with Upregulation of Proinflammatory Cytokine and Chemokine and Alpha Defensin Gene Expression in Colorectal Mucosa.

HIV may induce gastrointestinal (GI) mucosal immune dysregulation similar to inflammation observed in ulcerative colitis (UC). Colorectal biopsies from healthy controls (N=12) and from participants with HIV (N=20) or UC (N=9) were subjected to real time (RT)-PCR for selected cytokines, chemokines, antimicrobial peptides, Toll-like receptors, and inflammatory signaling and epithelial barrier proteins. HIV long terminal repeat relative copy number (RCN) in HIV participant biopsies was quantified by RT-PCR. Mean interleukin (IL)-6 mRNA levels did not differ significantly between HIV and UC participants (p=0.48) but were significantly higher relative to control mRNA levels only for HIV participants (p=0.03). Mean IL-8 and human defensin (HD) 5 mRNA levels were similar between HIV and UC participants (p=1.0 and p=0.35, respectively) and were significantly greater in both groups relative to controls (p<0.05 for all). Human beta-defensin (HBD)-2 mRNA levels were higher in UC relative to HIV and control participants (p<0.01 for both). Conversely, HBD-1 mRNA levels were downregulated in UC vs. HIV participants (p=0.01). Mediator gene expression did not differ significantly between HIV participants with detectable (N=10) or nondetectable (N=10) plasma viral loads. Tissue HIV relative copy number (RCN) correlated with plasma viral load (r=0.88, p<0.01) but not with mediator mRNA levels. The results of this study indicate that both chronic HIV infection and UC are associated with similar patterns of IL-6, IL- 8, and HD5 expression in colorectal biopsy tissue. These findings suggest overlapping mechanisms for GI mucosal inflammation in these two illnesses and merit further investigation in larger studies.

Regulation of human enteric α-defensins by NOD2 in the Paneth cell lineage.

Human enteric α-defensins (HD5 and HD6), major antimicrobial peptides produced by Paneth cells in the intestine, play important roles in intestinal innate immunity. Since their expression is decreased in Crohn's disease (CD), with decreased expression being more pronounced in the presence of NOD2 mutations, it would be extremely interesting to investigate the mechanism by which NOD2 may regulate expression of human enteric α-defensins. Here we show that although NOD2 by itself can slightly up-regulate expression of enteric α-defensins mainly through activation of the NF-κB pathway, it can strongly down-regulates their expression during differentiation of the Paneth cell lineage mainly by inhibiting activation of the MAPK pathway. Since NOD2 is over-expressed in CD and mutant NOD2 cannot result in NF-κB activity, our finding can provide an explanation of the previous observation showing decreased expression of human enteric α-defensin in CD and even more so in the presence of NOD2 mutations. In addition, this finding provides a new view on the function of NOD2 in regulating intestinal innate immunity.

Increased intestinal production of α-defensins in aged rats with acute pancreatic injury.

Acute pancreatitis is a life-threatening situation, frequently associated with uncontrolled local and systemic inflammation, and aging is associated with a worst prognosis. Antimicrobial peptides are ancient molecules that belong to innate immunity, produced by epithelial and immune cells, and are able to trigger a myriad of effector responses. We have hypothesized that antimicrobial peptides could play an important role during serious pancreatic injury. To investigate our hypothesis, α-defensin-5, α-defensin-7 and CRAMP gene expression levels were measured in the intestinal tissue of old and young rats submitted to chemical pancreatic damage. We found significantly higher levels of α-defensin-5 and α-defensin-7, but not CRAMP, in the samples from old mice. This increase was not associated with a worse systemic inflammatory response. We conclude that α-defensins may have a pivotal role during acute pancreatitis and that the elderly develops a more severe local, but not systemic inflammatory process.

Altered α-defensin 5 expression in cervical squamocolumnar junction: implication in the formation of a viral/tumour-permissive microenvironment.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix, which mainly develops at the squamocolumnar (SC) junction. The progression of cervical HPV infections into (pre)neoplastic lesions suggests that viral antigens are not adequately recognized by innate immunity or presented to the adaptive immune system. Members of the defensin family have recently been found to inhibit viral and bacterial pathogens, to stimulate the migration of immune cells and to play a role in anticancer responses. In the present study, we focused on the poorly characterized human α-defensin 5 (HD-5) and its possible role in these processes. We showed that HD-5 was able to prevent HPV virion entry into cervical keratinocytes and to influence adaptive immunity. Indeed, this peptide specifically induced the chemoattraction and proliferation of both activated T lymphocytes and immature dendritic cells in a CCR2/CCR6-dependent manner and stimulated the infiltration of these professional antigen-presenting cells in a (pre)neoplastic epithelium transplanted in vivo in immunodeficient mice. No chemotactic effect was observed with plasmacytoid dendritic cells, macrophages or natural killer cells. Proliferative and angiogenic effects of HD-5 were also assessed in vitro and in vivo. However there was a striking regional disparity in expression of HD-5, being prominent in ectocervical, vaginal and vulvar neoplasia, while absent, or nearly so, in the cervical SC junction. Taken together, these results suggest one possible explanation for why the SC junction is uniquely vulnerable to both high-risk HPV infection (via reduced HD-5 expression and viral entry) and progression of neoplasia (via altered cell-mediated immune responses and altered microenvironment).

Human α-defensin expression is not dependent on CCAAT/enhancer binding protein-ε in a murine model.

Specific granule deficiency (SGD) is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-ε (C/EBP-ε) gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore, azurophil granules contain diminished amounts of their most abundant proteins, α-defensins, also known as human neutrophil peptides (HNPs). In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-ε. Since mice do not express myeloid defensins, they cannot per se be used to characterize the role of C/EBP-ε in controlling HNP expression in vivo. We therefore crossed a transgenic HNP-1-expressing mouse with the Cebpe-/- mouse to study the in vivo significance of C/EBP-ε for HNP-1 transcription and expression. Surprisingly, neither expression nor processing of HNP-1 was affected by lack of C/EBP-ε in these mice. Transduction of C/EBP-ε into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern equivalent to endogenous secondary granule proteins. This limits the use of these transgenic mice as a model for human conditions.

Intratumoral expression of mature human neutrophil peptide-1 potentiates the therapeutic effect of doxorubicin in a mouse 4T1 breast cancer model.

Human neutrophil peptides (HNPs) 1-3 possess a high degree of similarity and are deregulated in many types of human tumors. Previous studies have demonstrated that tumor cell lines and microdissected fresh tumor cells express HNP1-3. In vitro, HNP1-3 have been reported to be cytotoxic to various types of tumor cells. Previously, we observed that the intracellular expression of HNP1 increased plasma membrane permeability in A549 cells and inhibited in vivo tumor angiogenesis. Based on these findings, we inferred that the intratumoral expression of HNP1 may benefit chemotherapy in solid tumors. In the present study, we established a mouse 4T1 breast cancer model imitating locally advanced breast cancer (LABC) and we injected the mice intratumorally with plasmid HNP1 (pHNP1). Doxorubicin (Dox) was administered twice i.v. at 5 mg/kg body weight on day 1 and 8. The possible mechanisms were investigated by evaluating cell proliferation and apoptosis, intracellular Dox accumulation, mitochondrial transmembrane potential (ΔΨm) and ultrastructural alteration of intratumoral microvessels. Compared with the single agent treatment, the combination of Dox and pHNP1 resulted in a significant delay in in vivo tumor growth and a decrease in lung metastasis. This chemosensitization effect was also observed in an A549 lung cancer model treated with cisplatin (DDP) and pHNP1. MTT assay and Annexin V staining demonstrated that 4T1 cells pre-transfected with pHNP1 were significantly more sensitive to Dox, causing increased proliferation inhibition and apoptosis. Further investigations showed that the intracellular expression of HNP1 enhanced Dox accumulation and significantly impaired mitochondrial ΔΨm. Moreover, in tumor tissues, HNP1 mediated intratumoral microvessel normalization and caused significant in situ tumor cell apoptosis. Our study suggests that intratumoral expression of HNP1 can significantly improve the therapeutic efficacy of Dox in breast cancer, abrogate the influence of multidrug resistance and enhance medication safety. Our findings may be important for the clinical therapeutic strategies of cancer chemotherapy.

Impaired immune defense in hemodialysis patients: role of α-defensins?

The mechanisms underlying the impaired immune response in hemodialysis (HD) patients are not completely understood. The α-defensins human neutrophil peptides-1, 2, and 3 are low molecular weight peptides with antimicrobial activity and important effector molecules of innate immune responses. We now examined the expression of these peptides in HD patients. Seventy-six patients on chronic HD treatment (mean time on HD 5.8 years; mean age 70 years) were studied and compared with 38 healthy volunteers and 20 patients with infections and normal renal function. Expression of α-defensins was analyzed semiquantitatively in leukocytes on the messenger RNA (mRNA) level by reverse transcriptase polymerase chain reaction; the α-defensin protein levels in serum were detected by enzyme-linked immunosorbent assay. α-Defensin concentrations (140 ± 10.5 ng/mL; mean ± standard error of the mean) as well as mRNA levels in leukocytes (82.9 ± 7.9 arbitrary units [a.u.]) in HD patients were not significantly different from those in healthy volunteers (156 ± 15.2 ng/mL; 81.4 ± 11.3 a.u.). Defensin levels were independent of the time of the patient on HD and their age. During infection periods (mean increase of the C-reactive protein to 161 ± 17.3 mg/L), defensin serum levels increased to 321 ± 65 ng/mL (P < 0.005) and mRNA expression in leukocytes to 159 ± 19.2 a.u. (P < 0.05). These increases were not significantly different from those in patients with normal renal function (298 ± 46.8 ng/mL and 128 ± 9.1 a.u., respectively) suffering from infections (C-reactive protein 222 ± 26.6 mg/L). Our results suggest that the impaired immune defense in dialysis patients is not due to a deficiency in α-defensins in these patients as neither basal levels nor expression during infections were reduced compared with subjects with normal renal function.

Macaque paneth cells express lymphoid chemokine CXCL13 and other antimicrobial peptides not previously described as expressed in intestinal crypts.

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), ß-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.

Intestinal Lin- c-Kit+ NKp46- CD4- population strongly produces IL-22 upon IL-1ß stimulation.

Small intestinal innate lymphoid cells (ILCs) regulate intestinal epithelial cell homeostasis and help to prevent pathogenic bacterial infections by producing IL-22. In a global gene-expression analysis comparing small intestinal ILCs (Lin(-)c-Kit(+)Sca-1(-) cells) with non-ILCs (Lin(-)c-Kit(-)Sca-1(-) cells), we found that Lin(-)c-Kit(+)Sca-1(-) cells highly expressed the mRNAs for Il22, antimicrobial peptides, Csf2rb2 (Il3r), mast cell proteases, and Rorc. We then subdivided the Lin(-)c-Kit(+)Sca-1(-) cells into three groups--Lin(-)c-Kit(+)NKp46(-)CD4(-), Lin(-)c-Kit(+)NKp46(-)CD4(+) (CD4(+) LTi-like cells), and Lin(-)c-Kit(+)NKp46(+) (NKp46(+) ILC22 cells)--and showed that the Lin(-)c-Kit(+)NKp46(-)CD4(-) cells produced the highest level of IL-22 protein after IL-1ß, IL-23, or IL-1ß and IL-23 stimulation. In addition, we showed that the majority of the Lin(-)c-Kit(+)NKp46(-)CD4(-) population was IL-7Rα(+)CD34(-)ß7(int) cells, and IL-7Rα(-) cells could be divided into three subsets (CD34(+)ß7(int), CD34(-)ß7(int), and CD34(int)ß7(hi) cells). The IL-7Rα(+)CD34(-)ß7(int) cells strongly expressed the transcripts for Il17f and Il22 after costimulation with IL-1ß and IL-23. The IL-7Rα(-)CD34(+)ß7(int) and IL-7Rα(-)CD34(int)ß7(hi) cells predominantly expressed the transcripts for mast cell proteases and differentiated almost entirely into mast cells after 1 wk in culture medium supplemented with a cytokine mixture, whereas the IL-7Rα(-)CD34(-)ß7(int) cells highly expressed α-defensins and showed no differentiation. Taken together, these findings indicate that the IL-7Rα(-)CD34(+)ß7(int) and IL-7Rα(-)CD34(int)ß7(hi) populations are mast cell progenitors, and the IL-7Rα(+)CD34(-)ß7(int) (CD4(-) LTi-like cells) and IL-7Rα(-)CD34(-)ß7(int) populations within Lin(-)c-Kit(+)NKp46(-)CD4(-) cells may control intestinal homeostasis and provide intestinal protection by producing high levels of IL-22 and α-defensins, respectively.