Lymph nodes (LNs) are integral sites for the generation of immune tolerance, migration of CD4 + T cells, and induction of Tregs. Despite the importance of LNs in regulation of inflammatory responses, the LN-specific factors that regulate T cell migration and the precise LN structural domains in which differentiation occurs remain undefined. Using intravital and fluorescent microscopy, we found that alloreactive T cells traffic distinctly into the tolerant LN and colocalize in exclusive regions with alloantigen-presenting cells, a process required for Treg induction. Extracellular matrix proteins, including those of the laminin family, formed regions within the LN that were permissive for colocalization of alloantigen-presenting cells, alloreactive T cells, and Tregs. We identified unique expression patterns of laminin proteins in high endothelial venule basement membranes and the cortical ridge that correlated with alloantigen-specific immunity or immune tolerance. The ratio of laminin α4 to laminin α5 was greater in domains within tolerant LNs, compared with immune LNs, and blocking laminin α4 function or inducing laminin α5 overexpression disrupted T cell and DC localization and transmigration through tolerant LNs. Furthermore, reducing α4 laminin circumvented tolerance induction and induced cardiac allograft inflammation and rejection in murine models. This work identifies laminins as potential targets for immune modulation.

Obesity has been associated with greater severity of influenza virus infection and impaired host defense. Exercise may confer health benefits even when weight loss is not achieved, but it has not been determined if regular exercise improves immune defense against influenza A virus (IAV) in the obese condition. In this study, diet-induced obese mice and lean control mice exercised for eight weeks followed by influenza viral infection. Exercise reduced disease severity in both obese and non-obese mice, but the mechanisms differed. Exercise reversed the obesity-associated delay in bronchoalveolar-lavage (BAL) cell infiltration, restored BAL cytokine and chemokine production, and increased ciliary beat frequency and IFNα-related gene expression. In non-obese mice, exercise treatment reduced lung viral load, increased Type-I-IFN-related gene expression early during infection, but reduced BAL inflammatory cytokines and chemokines. In both obese and non-obese mice, exercise increased serum anti-influenza virus specific IgG2c antibody, increased CD8+ T cell percentage in BAL, and reduced TNFα by influenza viral NP-peptide-responding CD8+ T cells. Overall, the results suggest that exercise “restores” the immune response of obese mice to a phenotype similar to non-obese mice by improving the delay in immune activation. In contrast, in non-obese mice exercise treatment results in an early reduction in lung viral load and limited inflammatory response.

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Background Trafficking and differentiation of naïve CD4+ and regulatory T cells (Treg) within the lymph node (LN) are integral for tolerance induction. The LN is comprised of stromal fibers that dictate lymphocyte migration and LN structure, organization, and microanatomic domains. Distribution of the stromal fiber ER-TR7 changes within the LN following antigenic challenge, but the contributions of ER-TR7 to the resulting immune response remain undefined. We hypothesized that these stromal fiber structural changes affect T cell fate and subsequently allograft survival. Methods C57BL/6 mice were left naïve (untreated) or made immune or tolerant (donor-specific BALB/c splenocyte transfusion −/+ anti-CD40L mAb), or made tolerant and received anti-ER-TR7 mAb. Donor-specific T cell migration was visualized by adoptive transfer of CFSE-labeled TEa T cell receptor transgenic CD4+ cells. Immunohistochemistry was performed on lymph nodes to detect stromal fiber distribution, structure, CCL21 presence, and Treg and donor-specific cell location relative to high endothelial venules (HEV). Naïve, tolerant, and tolerant + anti-ER-TR7 mice received BALB/c heterotopic cardiac allografts and graft survival was monitored. Results ER-TR7 distribution changed following the induction of tolerance vs. immunity. Treating tolerant mice with anti-ER-TR7 altered HEV basement membrane structure and the distribution of CCL21 within the LN. These differences were mirrored by changes in the migration of naïve and Treg cells within and surrounding the HEV. Anti-ER-TR7 prevented tolerance induction and resulted in allograft inflammation and rejection. Conclusions These results identify ER-TR7 as an important component of LN structure in tolerance and a direct target for immune modulation.

BackgroundAgriculture organic dust exposures induce lung disease with lymphoid aggregates comprised of both T and B cells. The precise role of B cells in mediating lung inflammation is unknown, yet might be relevant given the emerging role of B cells in obstructive pulmonary disease and associated autoimmunity.MethodsUsing an established animal model, C57BL/6 wild-type (WT) and B-cell receptor (BCR) knock-out (KO) mice were repetitively treated with intranasal inhalation of swine confinement organic dust extract (ODE) daily for 3 weeks and lavage fluid, lung tissues, and serum were collected.ResultsODE-induced neutrophil influx in lavage fluid was not reduced in BCR KO animals, but there was reduction in TNF-α, IL-6, CXCL1, and CXCL2 release. ODE-induced lymphoid aggregates failed to develop in BCR KO mice. There was a decrease in ODE-induced lung tissue CD11c+CD11b+ exudative macrophages and compensatory increase in CD8+ T cells in lavage fluid of BCR KO animals. Compared to saline, there was an expansion of conventional B2-, innate B1 (CD19+CD11b+CD5+/−)-, and memory (CD19+CD273+/-CD73+/−) B cells following ODE exposure in WT mice. Autoreactive responses including serum IgG anti-citrullinated protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) autoantibodies were increased in ODE treated WT mice as compared to saline control. B cells and serum immunoglobulins were not detected in BCR KO animals.ConclusionsLung tissue staining for citrullinated and MAA modified proteins were increased in ODE-treated WT animals, but not BCR KO mice. These studies show that agriculture organic dust induced lung inflammation is dependent upon B cells, and dust exposure induces an autoreactive response.Electronic supplementary materialThe online version of this article (10.1186/s12931-017-0703-x) contains supplementary material, which is available to authorized users.

Edited by Luke O'NeillLung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4R␣, and ␥C after IL-4 stimulation. Both IL-4R␣ and ␥C were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.Both genetic and environmental factors influence the onset and exacerbation of allergic asthma, which remains one of the most costly, non-communicable diseases of the human population. Allergic inflammation is characterized by airway hyperreactivity, type 2 cytokines (IL-4, -5, and -13), IgE, and the overproduction of mucus in the lung (1-3). Macrophages present at the site of allergic inflammation can be polarized by Th2-cytokines to become "alternatively activated" or M2 macrophages. In addition, the abundance of M2 macrophages in the lungs of asthmatics correlates with symptom severity (4 -6). As M2 macrophage polarization is dependent upon IL-4 stimulation, this cytokine and its signaling pathways represent excellent targets for asthma therapies.Our previous work has identified IL-4 signaling through the type I IL-4 receptor complex and activation of insulin receptor substrate 2 (IRS-2) 2 as important for the degree of polarization of M2 macrophages (7). IL-4 binds first to the IL-4R␣ chain with high affinity followed by recruitment of the common ␥ chain (IL-2R␥ or ␥C) to create a functional type I receptor. Assembly of this receptor complex leads to recruitment and phosphorylation of the JAK1 and -3 proteins followed by robust, yet transient, tyrosine phosphorylation of IRS-2 and STAT6. Phosphorylated STAT6 homodimerizes and translocates to the nucleus, ...

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

Aims:  The objective of this study was to estimate UV254 inactivation constants for four viral pathogens: influenza virus type A, porcine respiratory and reproductive syndrome virus (PRRSV), bovine viral diarrhoea virus (BVDV) and reovirus. Methods and Results:  Viruses in culture medium were exposed to one of nine doses of UV254 and then titrated for infectious virus. Analysis showed that viral inactivation by UV254 was more accurately described by a two‐stage inactivation model vs a standard one‐stage inactivation model. Conclusions:  The results provided evidence for the existence of two heterogeneous viral subpopulations among the viruses tested, one highly susceptible to UV254 inactivation and the other more resistant. Importantly, inactivation constants based on the one‐stage inactivation model would have underestimated the UV254 dose required for the inactivation of these viruses under the conditions of the experiment. Significance and Impact of the Study:  To improve the accuracy of estimates, it is recommended that research involving the inactivation of micro‐organisms evaluates inactivation kinetics using both one‐stage and two‐stage models. These results will be of interest to persons responsible for microbial agents under laboratory or field conditions.