Bronchus-associated Lymphoid Tissue Formation Research Articles

Smoking exposure-induced bronchus-associated lymphoid tissue in donor lungs does not prevent tolerance induction after transplantation

The presence of bronchus-associated lymphoid tissue (BALT) in donor lungs has been suggested to accelerate graft rejection after lung transplantation. Although chronic smoke exposure can induce BALT formation, the impact of donor cigarette use on alloimmune responses after lung transplantation is not well understood. Here, we show that smoking-induced BALT in mouse donor lungs contains Foxp3+ T cells and undergoes dynamic restructuring after transplantation, including recruitment of recipient-derived leukocytes to areas of pre-existing lymphoid follicles and replacement of graft-resident donor cells. Our findings from mouse and human lung transplant data support the notion that a donor’s smoking history does not predispose to acute cellular rejection or prevent the establishment of allograft acceptance with comparable outcomes to nonsmoking donors. Thus, our work indicates that BALT in donor lungs is plastic in nature and may have important implications for modulating proinflammatory or tolerogenic immune responses following transplantation.

Formation of bronchus-associated lymphoid tissue (BALT) for early life immune protection.

Formation of bronchus-associated lymphoid tissue (BALT) for early life immune protection.

Prior infection exposure alters the immune response at homeostasis and following SARS-CoV-2 vaccination and experimental infection in laboratory mice.

Abstract Variable translatability represents a major criticism of the use of mouse models for preclinical research. Lack of exposure to most endogenous murine pathogens results in natural immune responses of specific pathogen free (SPF) lab mice to more closely resemble that of newborns rather than adult humans. Recently, the effects of prior infection exposure (PIE) on immune maturation and responses have been increasingly explored in mice. Here, we employed a PIE model where BALB/c mice underwent sequential subclinical infection with four pathogens at two-week intervals: 1) MHV-68, a gammaherpesvirus, 2) MCMV, a betaherpesvirus, 3) influenza virus, and 4) Heligmosomoides polygyrus, a murine intestinal nematode. Four-to-12 weeks following H. polygyrus infection, mice were sacrificed, spleens were collected for flow cytometric analysis, and lungs were collected for histological evaluation. Compared to SPF mice, PIE spleens demonstrated increased proportions of CD4+ and CD8+ T effector memory cells. In the lungs, PIE induced the formation of bronchus-associated lymphoid tissue and increased infiltration of CD4+ and CD8+ T cells. Following vaccination against SARS-CoV-2 with either a protein subunit-based (S2P) or inactivated (iCoV2) vaccine, iCoV2-, but not S2P-, vaccinated PIE mice demonstrated significantly reduced serum neutralizing antibodies compared to SPF mice. Following infection with SARS-CoV-2-MA10, PIE mice showed a mild reduction in mortality, weight loss, and lung titers, and a significant increase in lung T cell infiltration compared to SPF mice. These data indicate that PIE results in adaptive immune maturation but has a variable effect on SARS-CoV-2-specific vaccine responses and clinical outcomes in mice. This work was supported by the NIH (K01OD026529, T32AI007151, U19AI100625, U19AI09680), an AAI Careers in Immunology Fellowship Award, an UNC School of Medicine Junior Investigator Development Award, and a NCTraCS and Emerging Challenges in Biomedical Research COVID Pilot Award.

Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection.

Respirovirus such as influenza virus infection induces pulmonary anti-viral immune response, orchestration of innate and adaptive immunity restrain viral infection, otherwise causes severe diseases such as pneumonia. Chemokines regulate leukocyte recruitment to the inflammation site. One chemokine CXCL5, plays a scavenging role to regulate pulmonary host defense against bacterial infection, but its role in pulmonary influenza virus infection is underdetermined. Here, using an influenza (H1N1) infected CXCL5-/- mouse model, we found that CXCL5 not only responds to neutrophil infiltration into infected lungs at the innate immunity stage, but also affects B lymphocyte accumulation in the lungs by regulating the expression of the B cell chemokine CXCL13. Inhibition of CXCL5-CXCR2 axis markedly induces CXCL13 expression in CD64+CD44hiCD274hi macrophages/monocytes in infected lungs, and in vitro administration of CXCL5 to CD64+ alveolar macrophages suppresses CXCL13 expression via the CXCL5-CXCR2 axis upon influenza challenge. CXCL5 deficiency leads to increased B lymphocyte accumulation in infected lungs, contributing to an enhanced B cell immune response and facilitating induced bronchus-associated lymphoid tissue formation in the infected lungs during the late infection and recovery stages. These data highlight multiple regulatory roles of CXCL5 in leukocyte chemotaxis during pulmonary influenza infection.

Intranasal inoculation of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia disease.

We evaluate the efficacy of recombinant DNA vaccine ABA392 against haemorrhagic septicaemia infection through intranasal administration route by targeting the mucosal immunity. The DNA vaccine was constructed and subjected to animal study using the Sprague Dawley (SD) rat. The study was divided into two major parts: (i) active and (ii) passive immunization studies, involving 30 animals for each part. Each group was then divided into five test groups: two test samples G1 and G2 with 50 and 100µgml-1 purified DNA vaccine; one positive control G5 with 106 CFU per ml formalin-killed PMB2; and two negative controls, G3 and G4 with normal saline and pVAX1 vector. Both studies were conducted for the determination of immunogenicity by total white blood cell count (TWBC), indirect ELISA and histopathological changes for the presence of the bronchus-associated lymphoid tissue (BALT). Our findings demonstrate that TWBC, IgA and IgG increased after each of the three vaccination regimes: groups G1, G2 and G5. Test samples G1 and G2 showed significant differences (P<0·05) compared to the negative controls, G3 and G4, but no significant differences from the positive control G5. Groups G1, G2 and G5 showed more formation of BALT compared to the negative controls, G3 and G4. Our results show that intranasal inoculation of recombinant DNA vaccine ABA392 can provoke mucosal immunity which makes it a potential prophylactic against HS. SIGNIFICANCE AND IMPACT OF THE STUDY: New approach of combating haemorrhagic septicaemia disease among bovines by recombinant DNA vaccine is crucial to overcome the loss of edible products from the infected bovines. DNA vaccine can potentially serve as a better immunogen which would elicit both cellular and humoral immunity, and it is also stable for its molecular reproduction. This research report demonstrates an effective yet simple way of administering the DNA vaccine via the intranasal route in rats, to provoke the mucosal immunity through the development of immunoglobulins IgA, IgG and bronchus-associated lymphoid tissue which guard as the first-line defence at the host's mucosal lining.

IL-22 Regulates Induction of Foxp3-Rich Tertiary Lymphoid Organs in Tolerant Lung Allografts

Purpose Intragraft tertiary lymphoid organs (TLOs) are generally associated with the generation of deleterious immune responses. In contrast, we have shown that Foxp3+ cells residing within bronchus-associated lymphoid tissue (BALT), a pulmonary TLO, are required for maintenance of allograft tolerance. Here, we set out to define the cellular and molecular mechanisms that mediate the induction of BALT in tolerant lung allografts. Methods Balb/c lungs were transplanted into B6 wild-type (WT), RORγt-Cre AHR flox (Group 3 innate lymphoid cells (ILC3)-deficient), TCRδ−/− (γδ T cell-deficient), anti-IL23 antibody-treated mice and IL-22 KO mice (n≥4/group). Recipients received anti-CD40L (day 0) and CTLA4-Ig (day 2) to induce tolerance. 7 or 30 days after transplantation grafts were evaluated by histology, flow cytometry and RT-PCR. Results 30 days after transplantation into WT hosts BALT was present in all lung allografts, characterized by expression of PNAd, a marker of high endothelial venules, and lymphoid follicles rich in B cells and Foxp3+ cells (Fig. 1). Graft expression of IL-22 and IL-23 (p Conclusion IL-22 regulates the formation of BALT within tolerant lungs through a process mediated by graft-infiltrating ILC3s and γδ T cells. This is in contrast to previous findings that induction of TLOs is dependent on lymphotoxin or IL-17 signaling. Thus, our findings have important implications for strategies to induce lung transplant tolerance.

The ABA392/pET30a protein of Pasteurella multocida provoked mucosal immunity against HS disease in a rat model

Haemorrhagic septicaemia (HS) is a well-known high fatality septicaemic disease happening among bovines. The disease is caused by the Pasteurella multocida serotype B:2 bacteria. P. multocida B:2 has high mortality and morbidity rates and is spread through the intranasal and oral routes in bovines. In this study, our aim was to investigate the efficacy of the recombinant protein vaccine, ABA392/pET30a via intranasal inoculation by targeting the mucosal immunity. The constructed recombinant protein vaccine ABA392/pET30a was subjected to an animal study using Sprague Dawley rats. The study was divided into two parts: active and passive immunization studies. Both studies were carried out through the determination of immunogenicity (using Total White Blood Cell (TWBC) Count with Indirect ELISA) and histopathogenicity, analyzing (Bronchus Associated Lymphoid Tissue (BALT) formation) in lungs. As a result, the IgA and IgG development of both tested groups: group 1 (50μg/mL protein vaccine) and group 2 (100μg/mL protein vaccine) showed equivalent with the positive control group 4 (formalin-killed P. multocida B:2). However, there was a significant difference when compared with the negative control group 3 (normal saline). These results demonstrate that both the protein vaccine at the concentration 50μg/mL and 100μg/mL have the same efficacy as the commercially available positive control vaccine. From the studies, higher concentration of protein vaccine at 100μg/mL showed higher development of both IgA and IgG compared to 50μg/mL protein vaccine. Higher and rapid development of IgA compared to IgG showed that mucosal immunity has been induced through the intranasal administration of the protein vaccine. In addition, leucocytosis was observed at each dose of vaccination showed that the protein vaccine is capable to induce the immune responses of the host. Histopathogenicity studies of the vaccinated groups showed more BALT formation and no severe lesions after challenge compared to the negative control group. Besides, no inflammatory onsite or anaphylactic responses were observed after the intranasal inoculation which proved to be safer and provided longer lasting immunity. Therefore, recombinant protein vaccine ABA392/pET30a could be a potential candidate for intranasal administration which can provoke mucosal immunity against HS disease.

Manifold Roles of CCR7 and Its Ligands in the Induction and Maintenance of Bronchus-Associated Lymphoid Tissue

The processes underlying the development and maintenance of tertiary lymphoid organs are incompletely understood. Using a Ccr7 knockout/knockin approach, we show that spontaneous bronchus-associated lymphoid tissue (BALT) formation can be caused by CCR7-mediated migration defects ofdendritic cells (DCs) in the lung. Plt/plt mice that lack the CCR7 ligands CCL19 and CCL21-serine do not form BALT spontaneously because lung-expressed CCL21-leucine presumably suffices to maintain steady-state DC egress. However, plt/plt mice are highly susceptible to modified vaccinia virus infection, showing enhanced recruitment of immune cells as well as alterations in CCR7-ligand-mediated lymphocyte egress from the lungs, leading to dramatically enhanced BALT. Furthermore, we identify two independent BALT homing routes for blood-derived lymphocytes. One is HEV mediated and depends on CCR7 and L-selectin, while the second route is via the lung parenchyma and is independent of these molecules. Together, these data provide insights into CCR7/CCR7-ligand-orchestrated aspects in BALT formation.

Pneumocystis-Driven Inducible Bronchus-Associated Lymphoid Tissue Formation Requires Th2 and Th17 Immunity

Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid structure composed of highly organized Tcell and B cell zones that forms in the lung in response to infectious or inflammatory stimuli. Here, we develop a model for fungal-mediated iBALT formation, using infection with Pneumocystis that induces development of pulmonary lymphoid follicles. Pneumocystis-dependent iBALT structureformation and organization required CXCL13 signaling. Cxcl13 expression was regulated by interleukin (IL)-17 family members, as Il17ra-/-, Il17rb-/-, and Il17rc-/- mice failed to develop iBALT. Interestingly, Il17rb-/- mice have intact Th17 responses, butfailed to generate an anti-Pneumocystis Th2 response. Given a role for Th2 and Th17 immunity iniBALT formation, we demonstrated that primary pulmonary fibroblasts synergistically upregulated Cxcl13 transcription following dual stimulation with IL-13 and IL-17A in a STAT3/GATA3-dependent manner. Together, these findings uncover a role for Th2/Th17 cells in regulating Cxcl13 expression and provide an experimental model for fungal-driven iBALT formation.

Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.

Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell- and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2(-/-)) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2(-/-) mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT-like lesion formation in the lungs of SE2(-/-) mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF-κB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expression in vivo These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation.-Solleti, S. K., Srisuma, S., Bhattacharya, S., Rangel-Moreno, J., Bijli, K. M., Randall, T. D., Rahman, A., Mariani, T. J. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.

Tertiary Lymphoid Structures Target the Antitumor Immune Response to Lung Cancer

Tertiary Lymphoid Structures Target the Antitumor Immune Response to Lung Cancer

IL-17–induced CXCL12 recruits B cells and induces follicle formation in BALT in the absence of differentiated FDCs

Ectopic lymphoid tissue, such as bronchus-associated lymphoid tissue (BALT) in the lung, develops spontaneously at sites of chronic inflammation or during infection. The molecular mechanisms underlying the neogenesis of such tertiary lymphoid tissue are still poorly understood. We show that the type of inflammation-inducing pathogen determines which key factors are required for the formation and maturation of BALT. Thus, a single intranasal administration of the poxvirus modified vaccinia virus Ankara (MVA) is sufficient to induce highly organized BALT with densely packed B cell follicles containing a network of CXCL13-expressing follicular DCs (FDCs), as well as CXCL12-producing follicular stromal cells. In contrast, mice treated with P. aeruginosa (P.a.) develop BALT but B cell follicles lack FDCs while still harboring CXCL12-positive follicular stromal cells. Furthermore, in IL-17-deficient mice, P.a.-induced BALT largely lacks B cells as well as CXCL12-expressing stromal cells, and only loose infiltrates of T cells are present. We show that Toll-like receptor pathways are required for BALT induction by P.a., but not MVA, and provide evidence that IL-17 drives the differentiation of lung stroma toward podoplanin-positive CXCL12-expressing cells that allow follicle formation even in the absence of FDCs. Taken together, our results identify distinct pathogen-dependent induction and maturation pathways for BALT formation.

Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1-enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C-producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.

The critical role of NADPH oxidase on inducible bronchus-associated lymphoid tissue formation in newborn mice

The critical role of NADPH oxidase on inducible bronchus-associated lymphoid tissue formation in newborn mice

Global Transcriptome Analysis in Influenza-Infected Mouse Lungs Reveals the Kinetics of Innate and Adaptive Host Immune Responses

An infection represents a highly dynamic process involving complex biological responses of the host at many levels. To describe such processes at a global level, we recorded gene expression changes in mouse lungs after a non-lethal infection with influenza A virus over a period of 60 days. Global analysis of the large data set identified distinct phases of the host response. The increase in interferon genes and up-regulation of a defined NK-specific gene set revealed the initiation of the early innate immune response phase. Subsequently, infiltration and activation of T and B cells could be observed by an augmentation of T and B cell specific signature gene expression. The changes in B cell gene expression and preceding chemokine subsets were associated with the formation of bronchus-associated lymphoid tissue. In addition, we compared the gene expression profiles from wild type mice with Rag2 mutant mice. This analysis readily demonstrated that the deficiency in the T and B cell responses in Rag2 mutants could be detected by changes in the global gene expression patterns of the whole lung. In conclusion, our comprehensive gene expression study describes for the first time the entire host response and its kinetics to an acute influenza A infection at the transcriptome level.

Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue

Presence and extent of bronchus-associated lymphoid tissue (BALT) is subject to considerable variations between species and is only occasionally observed in lungs of mice. Here we demonstrate that mice deficient for the chemokine receptor CCR7 regularly develop highly organized BALT. These structures were not present at birth but were detectable from day 5 onwards. Analyzing CCR7−/−/wild-type bone marrow chimeras, we demonstrate that the development of BALT is caused by alterations of the hematopoietic system in CCR7-deficient mice. These observations together with the finding that CCR7-deficient mice posses dramatically reduced numbers of regulatory T cells (T reg cells) in the lung-draining bronchial lymph node suggest that BALT formation might be caused by disabled in situ function of T reg cells. Indeed, although adoptive transfer of wild-type T reg cells to CCR7-deficient recipients resulted in a profound reduction of BALT formation, neither naive wild-type T cells nor T reg cells from CCR7−/− donors impair BALT generation. Furthermore, we provide evidence that CCR7-deficient T reg cells, although strongly impaired in homing to peripheral lymph nodes, are fully effective in vitro. Thus our data reveal a CCR7-dependent homing of T reg cells to peripheral lymph nodes in conjunction with a role for these cells in controlling BALT formation.

Frequency and potential cause of bronchus‐associated lymphoid tissue in fetal lungs

Bronchus-associated lymphoid tissue consists of lymphoid follicles with or without a germinal center within the bronchial wall. Bronchus-associated lymphoid tissue is part of the integrated mucosal immune system and present in about 50% of healthy infants. We examined a series of 141 fetal and neonatal lungs and detected bronchus-associated lymphoid tissue in 100% of cases with amniotic infection while postpartum perinatal pneumonia did not elicit bronchus-associated lymphoid tissue formation. Only rarely and in low density, bronchus-associated lymphoid tissue was present in non-infected fetuses. The in utero formation of bronchus-associated lymphoid tissue seems to be a reactive phenomenon and - as has been shown in another study - does not portend an adverse prognosis.