Cage Environment Regulates Gut Microbiota Independent of Toll-Like Receptors.

The gut microbiome orchestrates epithelial homeostasis and both local and remote immunological responses. Critical to these regulatory interactions are innate immune receptors termed Toll-like receptors (TLRs). Studies to date have implicated innate immunity and Toll-like receptors in shaping key features of the gut microbiome. However, a variety of biological and environmental variables are also implicated in determining gut microbiota composition. In this report, we hypothesized that cohousing and environment dominated the regulation of the gut microbiota in animal models independent of innate immunity. To determine the importance of these variables, innate immunity, or environment in shaping gut microbiota, we used a randomized cohousing strategy and transgenic TLR-deficient mice. We have found that mice cohoused together by genotype exhibited limited changes over time in the composition of the gut microbiota. However, for mice randomized to cage, we report extensive changes in the gut microbiota, independent of TLR function, whereby the fecal microbiota of TLR-deficient mice converges with that of wild-type mice. TLR5-deficient mice in these experiments exhibit greater susceptibility to comparative changes in the microbiota than other TLR-deficient mice and wild-type mice. Our work has broad implications for the study of innate immunity and host-microbiota interactions. Given the profound impact that gut dysbiosis may have on immunity, this report highlights the potential impact of cohousing on the gut microbiota and indices of inflammation as outcomes in biological models of infectious or inflammatory disease.

Bone marrow transplant-induced alterations in Notch signaling promote pathologic Th17 responses to γ-herpesvirus infection.

Idiopathic pneumonia syndrome (IPS) is a common, often fatal, complication following hematopoietic stem cell transplantation (HSCT) characterized by severe pneumonitis and interstitial fibrosis. Fully reconstituted syngeneic bone marrow transplant (BMT) mice infected with murine γ-herpesvirus-68 develop interleukin-17 (IL-17)-driven pneumonitis and fibrosis, which mimics clinical manifestations of IPS. We found CD103+ and CD11b+ dendritic cells (DCs) are selectively deficient for the Notch ligand, DLL4, following BMT and CD4+ T cells isolated from lungs and spleens of infected BMT mice display Notch signaling defects. Mice transplanted with CD4-Cre-driven dominant-negative Notch transcriptional regulator Mastermind-Like (CD4-Cre-DNMAML (CCD) mice) bone marrow displayed elevated IL-17 and transforming growth factor-ß (TGF ß) in the lungs, a further expansion of T-helper type 17 (Th17) cells, and developed more fibrosis than wild-type (WT)-BMT mice. Culture of BMT lung leukocytes with recombinant Notch ligand, DLL4, restored Notch signaling and decreased production of IL-17. Adoptive transfer of CD11c+ DCs could restore Th1 and limit Th17 in WT-BMT but not CCD-BMT mice, indicating that a specific DC/CD4+ T-cell Notch interaction modulates IL-17 production following reconstitution in syngeneic BMT mice. Given recent clinical observations showing that patients with pulmonary complications post-transplant harbor occult herpesvirus infections, these data provide mechanistic insight and suggest potential therapies for these devastating conditions.

Elevated prostaglandin E2 post-bone marrow transplant mediates interleukin-1ß-related lung injury.

Hematopoietic stem cell transplant (HSCT) treats or cures a variety of hematological and inherited disorders. Unfortunately, patients who undergo HSCT are susceptible to infections by a wide array of opportunistic pathogens. Pseudomonas aeruginosa bacteria can have life-threatening effects in HSCT patients by causing lung pathology that has been linked to high levels of the potent pro-inflammatory cytokine, interleukin-1ß (IL-1ß). Using a murine bone marrow transplant (BMT) model, we show that overexpression of prostaglandin E2 (PGE2) post-BMT signals via EP2 or EP4 to induce cyclic adenosine monophosphate (cAMP), which activates protein kinase A or the exchange protein activated by cAMP (Epac) to induce cAMP response element binding-dependent transcription of IL-1ß leading to exacerbated lung injury in BMT mice. Induction of IL-1ß by PGE2 is time and dose dependent. Interestingly, IL-1ß processing post-P. aeruginosa infection occurs via the enzymatic activity of either caspase-1 or caspase-8. Furthermore, PGE2 can limit autophagy-mediated killing of P. aeruginosa in alveolar macrophages, yet autophagy does not have a role in PGE2-mediated upregulation of IL-1ß. Reducing PGE2 levels with indomethacin improved bacterial clearance and reduced IL-1ß-mediated acute lung injury in P. aeruginosa-infected BMT mice.

Transbronchial biopsy in the management of pulmonary complications of hematopoietic stem cell transplantation.

The utility of transbronchial biopsy in the management of pulmonary complications following hematopoietic stem cell transplantation (HSCT) has shown variable results. Herein, we examine the largest case series of patients undergoing transbronchial biopsy following HSCT. We performed a retrospective analysis of 130 transbronchial biopsy cases performed in patients with pulmonary complications post HSCT. Logistic regression models were applied to examine diagnostic yield, odds of therapy change and complications. The most common histologic finding on transbronchial biopsy was a nonspecific interstitial pneumonitis (n=24 cases, 18%). Pathogens identified by transbronchial biopsy were rare, occurring in <5% of cases. A positive transbronchial biopsy significantly increased the odds of a subsequent change in corticosteroid therapy (odds ratio (OR)=3.12; 95% confidence interval (CI) 1.18-8.23; P=0.02) but was not associated with a change in antibiotic therapy (OR=1.01; 95% CI 0.40-2.54; P=0.98) or changes in overall therapy (OR=1.92; 95% CI 0.79-4.70; P=0.15). Patients who underwent a transbronchial biopsy had increased odds of complications related to the bronchoscopy (OR=3.33, 95% CI 1.63-6.79; P=0.001). In conclusion, transbronchial biopsy may contribute to the diagnostic management of noninfectious lung injury post HSCT, whereas its utility in the management of infectious pulmonary complications of HSCT remains low.

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

Periostin regulates fibrocyte function to promote myofibroblast differentiation and lung fibrosis.

Fibrocytes are circulating mesenchymal precursors (CD45+, col 1+) recruited to fibrotic areas. Fibrocytes secrete profibrotic mediators including periostin; a matricellular protein that regulates cellular interactions with extracellular matrix (ECM) components. In bleomycin-induced fibrosis, periostin deficiency in structural or hematopoietic cells limits development of pulmonary fibrosis. To determine if hematopoietic-derived fibrocytes might secrete soluble factors to activate structural myofibroblast differentiation, wild-type (WT) fibroblasts were treated with conditioned medium from fibrocytes isolated from bleomycin-treated WT or periostin-/- mice. After 24 h we saw less α-smooth muscle actin expression in cells treated with conditioned medium from periostin-/- fibrocytes. Adoptive transfer of WT fibrocytes augmented lung fibrosis to a greater extent than transfer of fibrocytes from periostin-/- mice. In vitro analysis of fibrocytes and fibroblasts isolated from WT and periostin-/- mice treated with TGFß1 or periostin demonstrated co-regulation of mesenchymal activation and beta 1 integrin as a potential receptor for periostin on fibrocytes. Additionally, connective tissue growth factor (CTGF) mRNA expression was increased in fibrocytes treated with periostin whereas CTGF and lysl oxidase (LOX) mRNA expression was low in bleomycin-treated periostin-/- fibrocytes. These data suggest fibrocytes may augment bleomycin-induced fibrosis via secretion of periostin and other soluble factors that promote myofibroblast differentiation.

IL-36γ is a crucial proximal component of protective type-1-mediated lung mucosal immunity in Gram-positive and -negative bacterial pneumonia.

Interleukin-36γ (IL-36γ) is a member of novel IL-1-like proinflammatory cytokine family that are highly expressed in epithelial tissues and several myeloid-derived cell types. Little is known about the role of the IL-36 family in mucosal immunity, including lung anti-bacterial responses. We used murine models of IL-36γ deficiency to assess the contribution of IL-36γ in the lung during experimental pneumonia. Induction of IL-36γ was observed in the lung in response to Streptococcus pneumoniae (Sp) infection, and mature IL-36γ protein was secreted primarily in microparticles. IL-36γ-deficient mice challenged with Sp demonstrated increased mortality, decreased lung bacterial clearance and increased bacterial dissemination, in association with reduced local expression of type-1 cytokines, and impaired lung macrophage M1 polarization. IL-36γ directly stimulated type-1 cytokine induction from dendritic cells in vitro in a MyD88-dependent manner. Similar protective effects of IL-36γ were observed in a Gram-negative pneumonia model (Klebsiella pneumoniae). Intrapulmonary delivery of IL-36γ-containing microparticles reconstituted immunity in IL-36γ-/- mice. Enhanced expression of IL-36γ was also observed in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome because of pneumonia. These studies indicate that IL-36γ assumes a vital proximal role in the lung innate mucosal immunity during bacterial pneumonia by driving protective type-1 responses and classical macrophage activation.

Focal interstitial CC chemokine receptor 7 (CCR7) expression in idiopathic interstitial pneumonia.

BACKGROUND/AIMS: Idiopathic interstitial pneumonias (IIPs) are a diverse grouping of chronic pulmonary diseases characterised by varying degrees of pulmonary fibrosis. The triggers of the fibroproliferative process in IIP remain enigmatic but recent attention has been directed towards chemokine involvement in this process. METHODS: The expression of two chemokine receptors, CCR7 and CXCR4, and their respective ligands, CCL19, CCL21, and CXCL12, were examined in surgical lung biopsies (SLBs) from patients with IIP. Transcript and protein expression of these receptors and their ligands was compared with that detected in histologically normal margin SLBs. RESULTS: CCR7 and CXCR4 were detected by gene array and real time polymerase chain reaction analysis and CCR7, but not CXCR4, expression was significantly raised in usual interstitial pneumonia (UIP) relative to biopsies from patients diagnosed with non-specific interstitial pneumonia (NSIP) or respiratory bronchiolitis/interstitial lung disease (RBILD). CCR7 protein was expressed in interstitial areas of all upper and lower lobe UIP SLBs analysed. CCR7 expression was present in 50% of NSIP SLBs, and CCR7 was restricted to blood vessels and mononuclear cells in 75% of RBILD SLBs. Immune cell specific CXCR4 expression was seen in IIP and normal margin biopsies. CCR7 positive areas in UIP biopsies were concomitantly positive for CD45 (the leucocyte common antigen) but CCR7 positive areas in all IIP SLBs lacked the haemopoietic stem cell antigen CD34, collagen 1, and alpha smooth muscle actin. CONCLUSION: This molecular and immunohistochemical analysis showed that IIPs are associated with abnormal CCR7 transcript and protein expression.

Bone marrow transplantation alters lung antigen-presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection.

Hematopoietic stem cell transplantation (HSCT) efficacy is limited by numerous pulmonary complications. We developed a model of syngeneic bone marrow transplantion (BMT) followed by infection with murine gamma herpesvirus-68 that results in pneumonitis and fibrosis and mimics human "noninfectious" HSCT complications. BMT mice experience increased early lytic replication, but establish viral latency by 21 days post infection. CD4 T cells in BMT mice are skewed toward interleukin (IL)-17A rather than interferon (IFN)-γ production. Transplantation of bone marrow from Il-17a(-/-) donors or treatment with anti-IL-17A neutralization antibodies at late stages attenuates pneumonitis and fibrosis in infected BMT mice, suggesting that hematopoietic-derived IL-17A is essential for development of pathology. IL-17A directly influences activation and extracellular matrix production by lung mesenchymal cells. Lung CD11c+ cells of BMT mice secrete more transforming growth factor beta-ß1, and pro-TH17 mRNAs for IL-23 and IL-6, and less TH1-promoting cytokine mRNA for IFN-γ but slightly more IL-12 mRNA in response to viral infection. Adoptive transfer of non-BMT lung CD11c-enriched cells restores robust TH1 response and suppresses aberrant TH17 response in BMT mice to improve lung pathology. Our data suggest that "noninfectious" HSCT lung complications may reflect preceding viral infections and demonstrate that IL-17A neutralization may offer therapeutic advantage even after disease onset.

The role of CXC chemokines in the regulation of angiogenesis in association with lung cancer.

Recent evidence demonstrates that members of the CXC chemokine family can act as either angiogenic or angiostatic factors, depending on the presence of the ELR (Glu-Leu-Arg) motif in their NH(2) terminus. As such, these molecules have been shown to regulate tumor growth and metastasis in an animal model of human non-small cell lung cancer. ELR-positive members (for example, IL-8) are tumor-derived proteins that promote tumor growth and metastasis. Conversely, ELR-negative members (for example, IP-10 and MIG) are endogenous factors that inhibit tumor growth and metastasis. The levels of these factors in human tumor specimens may have predictive value for determining which patients are at risk for developing metastasis, and alteration of these CXC chemokine levels may provide a therapeutic intervention.

Chronic reactive airway disease following acute chlorine gas exposure in an asymptomatic atopic patient.

While chlorine gas inhalation has previously been reported to cause temporary mucous membrane irritation, acute pneumonitis, pulmonary edema, and transient bronchospasm, there is controversy about the existence of long-term pulmonary sequelae. We report the case of a 25-year-old man in whom chronic, recurrent asthma developed after exposure to a chlorine gas leak in an enclosed space. His course since the exposure has been notable for frequent exacerbations necessitating chronic corticosteroid therapy and multiple hospitalizations. To our knowledge, the persistence of symptoms years after the exposure is unique in the literature.

The Managed Care Education Clearinghouse.

The authors surveyed all 125 allopathic medical schools to determine the number of schools that had implemented a formal curriculum in managed care and how many had a substantial interest in a Web-based clearinghouse for managed care curricular resources. They describe the results of their survey and the Web site they developed, the Managed Care Education Clearinghouse.

Neuralgia after inguinal hernia repair.

Severe chronic pain after groin hernia repair is uncommon but potentially debilitating. Fifteen patients with this condition were retrospectively reviewed. All patients had severe pain, which prevented their working or normal activity and was refractory to nonoperative treatment. Essentials of therapy included 1) a preoperative attempt to identify the involved nerve and 2) high ligation and division of the involved nerve identified at exploration. Twelve patients obtained excellent results and were able to return to normal activity with no requirement for analgesia. Understanding of the typical nerve anatomy, as well as the individual variation in nerve anatomy, can help prevent this complication and is essential for correction if the complication does develop.

Recombination potential of the human DIR elements.

The human DIR genes are putative DH elements that are GC rich and are found between DN and DM DH gene segments. The DIR genes each have consensus recombination signal sequences (RSS) that could conceivably generate DIR coding regions of about 126 nucleotides. These RSS should allow for DH-DH rearrangements that do not violate the 12/23 recombination rule. Several Ig CDR3 sequences have been assigned to DIR usage; however, there are frequent gaps and mismatches associated with these assignments. In some instances these CDR3 sequences might be better explained by GC rich N segment addition. This report analyzes the recombination potential of the human DIR elements by PCR. Though DH-JH rearrangement of the DH genes flanking the DIR regions (DM and DN) are easily demonstrated, very little evidence of DIR-JH rearrangement could be documented. Furthermore, in amplifications that should concurrently detect both DH-DIR rearrangements (which maintain the 12/23 recombination rule) and DH-DH rearrangements (which violate the 12/23 recombination rule), DH-DH rearrangements predominate. We conclude that the DIR-associated RSS participate minimally in both DH-DH and DH-JH rearrangement. In addition, we describe several conventional DH-DH rearrangement intermediates, demonstrating unequivocally that this phenomena occurs during human Ig rearrangement.

Role of T- and B-lymphocytes in pulmonary host defences.

Pulmonary infectious diseases cause significant morbidity and mortality in both industrialized and developing countries. Adaptive immune responses are required to defend the lung against pathogens that survive in normal macrophages and extracellular organisms that evade phagocytosis. Microbes initiate both innate immune responses and specific adaptive immune responses. Innate immune response molecules regulate T-lymphocyte differentiation. Activated T-lymphocytes provide cytokines, which activate macrophages and lytic signals that lyse infected antigen-presenting cells. Antibodies produced by plasma cells facilitate microbial clearance through diverse effector mechanisms including opsonization, complement fixation and antibody-dependent cytotoxicity. Lymphocytes determine the specificity of the immune response and orchestrate effector limbs of the immune response.

A community-based study of near-fatal asthma.

BACKGROUND: The purpose of this study was to describe the community-based impact of near-fatal asthma within the District of Columbia (Washington, DC). METHODS: The design was a prospective cohort study. Subjects included all persons in 1993 who presented to Washington, DC hospitals alive, requiring intubation for respiratory failure (including subjects who subsequently died in the hospital). Washington, DC hospitals were contacted on a biweekly basis to identify subjects. Patients were contacted by mail, followed by an interview with the subject or proxy. RESULTS: Of the 35 case subjects identified, 31 (88.6%) were interviewed. Sixty-one percent of the subjects were female; 84% were African-American; and 45.2% were less than 18 years old. Forty-five percent had asthma for 10 or more years. Twenty-three percent reported the emergency department as their usual source of health care, and 32% saw a provider on a weekly basis. Fifty-two percent were taking four or more prescription medications, and 29% were taking no anti-inflammatory medications. In the 24 hours before the event, 77% reported difficulty breathing, but only 64% reported contacting a health care provider. CONCLUSIONS: Community-based investigation of near-fatal asthma may lead to a better characterization of risk factors associated with this event. Findings from this study suggest that some of the factors associated with near-fatal events may be different from those associated with fatal asthma and that up to one third of the events may have been preventable.

Regulatory elements necessary for termination of transcription within the Ig heavy chain gene locus.

Previous experiments have shown that the extent of delta gene transcription during B cell development is regulated primarily at the transcriptional level. We have shown that deletion of a sequence located between the mu and delta coding regions in the Ig heavy chain locus where transcriptional termination has been previously mapped abrogates the termination. Restoration of termination requires reintroduction of this segment as well as sequence elements within the microM poly (A) site which cannot be substituted by the microS poly (A) site. Recognition of the termination site by non-lymphoid cells suggests that initiation of delta transcription in mature B lymphocytes requires the activation of an anti-termination mechanism not yet developed in early B cells.

Transcriptional analysis of inhibition of lipopolysaccharide response by anti-IgM.

Murine splenic B cells, when stimulated with LPS, show a generalized enhancement of gene transcription. In addition to this general increase, there is a specific increase in microseconds mRNA production and differentiation to high rate IgM secretion. Anti-mu added concomitantly with LPS at the start of culture has been demonstrated to inhibit the LPS-induced increase in microseconds mRNA production without affecting the proliferative capacity of the cells. By "run-on" analysis of nascent transcription, we have shown that the effect of anti-mu is mediated by the abrogation of the up-regulation of transcription of the mu-gene induced by LPS. Furthermore, by assessing the site of transcription termination, it is possible to infer that alterations in 3'-end processing induced by LPS are also inhibited. We have also found that CAT3 gene activity driven by a number of promoter/enhancers with diverse regulatory motifs are inhibited by anti-mu. These results suggest that the effect of anti-mu cannot be restricted to interactions with a single regulatory element. Therefore, cross-linking of surface IgM may affect a number of genes involved in differentiation to Ig secretion.

Immunoglobulin D(H) recombination signal sequence targeting: effect of D(H) coding and flanking regions and recombination partner.

V(D)J recombination is targeted by recombination signal sequences (RSS) located immediately adjacent to immune receptor gene segments. While the RSS flanking D(H) segments appear to be equivalent, they are not randomly utilized. During D(H) to J(H) rearrangement, the 3' D(H) RSS is virtually exclusively utilized, suggesting that the 3' D(H) RSS could simply be a better target for the recombinase. However, when we examined V(H) to D(H) (without J(H)) rearrangements, we found that the preference for D(H) RSS use changes, so that the 5' D(H) RSS are preferred. This suggests that the 3' D(H) RSS are not simply superior targets for the V(D)J recombinase, but instead that certain 12/23-bp spacer RSS combinations work better together to target recombination than do others. We have analyzed a series of artificial recombination substrates to delineate cis sequences that affect D(H) RSS selection. Our data suggest that coding sequences adjacent to the D(H) RSS, flanking sequences outside the D(H) gene segment itself, and recombination partner all affect D(H) RSS targeting.