Emerging role of immune cells as drivers of pulmonary fibrosis.

The pathogenesis of pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF) and other forms of interstitial lung disease, involves a complex interplay of various factors including host genetics, environmental pollutants, infection, aberrant repair and dysregulated immune responses. Highly variable clinical outcomes of some ILDs, in particular IPF, have made it difficult to identify the precise mechanisms involved in disease pathogenesis and thus the development of a specific cure or treatment to halt and reverse the decline in patient health. With the advent of in-depth molecular diagnostics, it is becoming evident that the pathogenesis of IPF is unlikely to be the same for all patients and therefore will likely require different treatment approaches. Chronic inflammation is a cardinal feature of IPF and is driven by both innate and adaptive immune responses. Inflammatory cells and activated fibroblasts secrete various pro-inflammatory cytokines and chemokines that perpetuate the inflammatory response and contribute to the recruitment and activation of more immune cells and fibroblasts. The balance between pro-inflammatory and regulatory immune cell subsets, as well as the interactions between immune cell types and resident cells within the lung microenvironment, ultimately determines the extent of fibrosis and the potential for resolution. This review examines the role of the innate and adaptive immune responses in pulmonary fibrosis, with an emphasis on IPF. The role of different immune cell types is discussed as well as novel anti-inflammatory and immunotherapy approaches currently in clinical trial or in preclinical development.

Immunological Phenotyping of Mice with a Point Mutation in Cdk4.

Cyclin-dependent kinases (CDKs) play a crucial role in regulation of the mammalian cell cycle. CDK4 and CDK6 control the G1/S restriction checkpoint through their ability to associate with cyclin D proteins in response to growth factor signals. CDK4 deficiency in mice gives rise to a range of endocrine-specific phenotypes including diabetes, infertility, dwarfism, and atrophy of the anterior pituitary. Although CDK6 deficiency can cause thymic atrophy due to a block in the double-negative (DN) to double-positive (DP) stage of T cell development, there are no overt defects in immune cell development reported for CDK4-deficient mice. Here, we examined the impact of a novel N-ethyl-N-nitrosourea-induced point mutation in the gene encoding CDK4 on immune cell development. Mutant mice (Cdk4wnch/wnch) showed normal development and differentiation of major immune cell subsets in the thymus and spleen. Moreover, T cells from Cdk4wnch/wnch mice exhibited normal cytokine production in response to in vitro stimulation. However, analysis of the mixed bone marrow chimeras revealed that Cdk4wnch/wnch-derived T cell subsets and NK cells are at a competitive disadvantage compared to Cdk4+/+-derived cells in the thymus and periphery of recipients. These results suggest a possible role for the CDK4wnch mutation in the development of some immune cells, which only becomes apparent when the Cdk4wnch/wnch mutant cells are in direct competition with wild-type immune cells in the mixed bone marrow chimera.

Exercise-induced responses in matrix metalloproteinases and osteopontin are not moderated by exercise format in males with overweight or obesity.

PURPOSE: Matrix metalloproteinase-2 (MMP-2) and -3 (MMP-3), and osteopontin (OPN) are associated with adipose-tissue expansion and development of metabolic disease. The purpose of the current study was to assess the circulating concentration of these markers, along with adiponectin and glucose concentrations, in response to acute exercise in individuals with overweight or obesity. METHODS: Fourteen sedentary males with overweight or obesity (29.0 ± 3.1 kg/m2) completed two separate, 3-day trials in randomised and counterbalanced order. An oral glucose tolerance test (OGTT) was performed on each day of the trial. Day two of each trial consisted of a single 30 min workload-matched bout of either high-intensity interval exercise (HIIE; alternating 100% and 50% of peak pulmonary oxygen uptake, [Formula: see text]O2peak) or continuous moderate intensity (CME; 60% [Formula: see text]O2peak) cycling completed 1 h prior to the OGTT. Glucose and physical activity were continuously monitored, while MMP-2, MMP-3, OPN and adiponectin were measured pre-, 0 h post-, 1 h post- and 25 h post-exercise. RESULTS: Exercise transiently increased MMP-3 and decreased OPN (both p < 0.01), but not MMP-2 or adiponectin. There were no differences in the response of inflammatory markers to the different exercise formats. Exercise increased mean daily glucose concentration and area under the glucose curve during the OGTT on Day 2 and Day 3 (main effect of time; p < 0.05). CONCLUSION: Acute cycling exercise decreased OPN, which is consistent with longer term improvements in cardiometabolic health and increased MMP-3, which is consistent with its role in tissue remodelling. Interestingly, exercise performed prior to the morning OGTT augmented the glucose concentrations in males. TRIAL REGISTRATION: ACTRN12613001086752.

Acute T-Cell-Driven Inflammation Requires the Endoglycosidase Heparanase-1 from Multiple Cell Types.

It has been accepted for decades that T lymphocytes and metastasising tumour cells traverse basement membranes (BM) by deploying a battery of degradative enzymes, particularly proteases. However, since many redundant proteases can solubilise BM it has been difficult to prove that proteases aid cell migration, particularly in vivo. Recent studies also suggest that other mechanisms allow BM passage of cells. To resolve this issue we exploited heparanase-1 (HPSE-1), the only endoglycosidase in mammals that digests heparan sulfate (HS), a major constituent of BM. Initially we examined the effect of HPSE-1 deficiency on a well-characterised adoptive transfer model of T-cell-mediated inflammation. We found that total elimination of HPSE-1 from this system resulted in a drastic reduction in tissue injury and loss of target HS. Subsequent studies showed that the source of HPSE-1 in the transferred T cells was predominantly activated CD4+ T cells. Based on bone marrow chimeras, two cellular sources of HPSE-1 were identified in T cell recipients, one being haematopoiesis dependent and the other radiation resistant. Collectively our findings unequivocally demonstrate that an acute T-cell-initiated inflammatory response is HPSE-1 dependent and is reliant on HPSE-1 from at least three different cell types.

An Outbreak of Highly Pathogenic Avian Influenza (H7N7) in Australia and the Potential for Novel Influenza A Viruses to Emerge.

In 2020, several geographically isolated farms in Victoria, Australia, experienced an outbreak of highly pathogenic avian influenza (HPAI) virus H7N7 and low pathogenic avian influenza (LPAI) viruses H5N2 and H7N6. Effective containment and control measures ensured the eradication of these viruses but the event culminated in substantial loss of livestock and significant economic impact. The avian HPAI H7N7 virus generally does not infect humans; however, evidence shows the ocular pathway presents a favourable tissue tropism for human infection. Through antigenic drift, mutations in the H7N7 viral genome may increase virulence and pathogenicity in humans. The Victorian outbreak also detected LPAI H7N6 in emus at a commercial farm. Novel influenza A viruses can emerge by mixing different viral strains in a host susceptible to avian and human influenza strains. Studies show that emus are susceptible to infections from a wide range of influenza viral subtypes, including H5N1 and the pandemic H1N1. The emu's internal organs and tissues express abundant cell surface sialic acid receptors that favour the attachment of avian and human influenza viruses, increasing the potential for internal genetic reassortment and the emergence of novel influenza A viruses. This review summarises the historical context of H7N7 in Australia, considers the potential for increased virulence and pathogenesis through mutations and draws attention to the emu as potentially an unrecognised viral mixing vessel.

Strength Training Improves Exercise Economy in Triathletes During a Simulated Triathlon.

PURPOSE: The completion of concurrent strength and endurance training can improve exercise economy in cyclists and runners; however, the efficacy of strength training (ST) implementation to improve economy in long-distance (LD) triathletes has not yet been investigated. The purpose of this study was to investigate physiological outcomes in LD triathletes when ST was completed concurrently to endurance training. METHODS: A total of 25 LD triathletes were randomly assigned to either 26 weeks of concurrent endurance and ST (n = 14) or endurance training only (n = 11). The ST program progressed from moderate (8-12 repetitions, ≤75% of 1-repetition maximum, weeks 0-12) to heavy loads (1-6 repetitions, ≥85% of 1-repetition maximum, weeks 14-26). Physiological and performance indicators (cycling and running economy, swim time, blood lactate, and heart rate) were measured during a simulated triathlon (1500-m swim, 60-min cycle, and 20-min run) at weeks 0, 14, and 26. Maximal strength and anthropometric measures (skinfolds and body mass) were also collected at these points. RESULTS: The endurance strength group significantly improved maximal strength measures at weeks 14 and 26 (P < .05), cycling economy from weeks 0 to 14 (P < .05), and running economy from weeks 14 to 26 (P < .05) with no change in body mass (P > .05). The endurance-only group did not significantly improve any economy measures. CONCLUSIONS: The addition of progressive load ST to LD triathletes' training programs can significantly improve running and cycling economy without an increase in body mass.

Strength Training in Long-Distance Triathletes: Barriers and Characteristics.

ABSTRACT: Luckin, KM, Badenhorst, CE, Cripps, AJ, Landers, GJ, Merrells, RJ, Bulsara, MK, and Hoyne, GF. Strength training in long-distance triathletes: Barriers and characteristics. J Strength Cond Res 35(2): 495-502, 2021-The purpose of this investigation was to identify perceived and physical barriers toward the completion of concurrent strength training and endurance training in long-distance triathletes. Three hundred ninety long-distance triathletes (224 women, 166 men; age [y]: 39 ± 10) completed a 68-question self-administered, semiquantitative survey that assessed endurance and strength training characteristics, experience in triathlon, and perceived barriers regarding the completion of strength training. Mean training hours per week was 14.92 ± 5.25, with 54.6% reporting participation in strength training. Heavy strength training was the most commonly reported (39.4%), with significantly more men completing this form of strength training (p < 0.001). Results from subjects who did not complete strength training indicated that perceived time constraints (53.1%) in addition to lack of knowledge on exercise progression and form (52.5%) are prominent perceived barriers to strength training completion. Identification of the barriers perceived by long-distance triathletes that prevent them from completing concurrent strength training and endurance training may be useful for coaches, athletes, and sports scientists who seek to incorporate strength training for injury prevention and performance improvement.

Loss of hnRNPLL-dependent splicing of Ptprc has no impact on B-cell development, activation and terminal differentiation into antibody-secreting cells.

The RNA-binding protein heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL) controls alternative splicing of protein tyrosine phosphatase receptor type C (Ptprc) which encodes CD45. hnRNPLL deficiency leads to a failure in silencing Ptprc exons 4-6 causing aberrant expression of the corresponding CD45 isoforms, namely, CD45RA, RB and RC. While an N-ethyl-N-nitrosourea-induced point mutation in murine Hnrnpll results in loss of peripheral naïve T cells, its role in B-cell biology remains unclear. Here, we demonstrate that B-cell development in the bone marrow of Hnrnpllthu/thu mice is normal and the number of mature B-cell subsets in the spleen and peritoneal cavity is comparable to control littermates. In response to in vivo immunization, Hnrnpllthu/thu mice were deficient in generating germinal center (GC) B cells, and analysis of mixed bone marrow chimeras revealed that the GC B-cell deficiency was a B-cell extrinsic effect of the hnRNPLL mutation. Mature Hnrnpllthu/thu B cells proliferated normally in response to various B-cell receptor- and Toll-like receptor-mediated stimuli. Similarly, in vitro stimulation of mutant B cells led to normal generation of plasmablasts, but mutant plasmablasts failed to downregulate B220 expression because of the inability of cells to undergo proper CD45 pre-messenger RNA alternative splicing. These findings collectively suggest that, like in T and natural killer T cells, the mutation disrupts hnRNPLL-mediated alternative splicing of the Ptprc gene in plasmablasts, but this dysregulation of Ptprc alternative splicing does not affect the development and function of B cells.

Behavioural phenotyping of thunder mice with a hypomorphic mutation of heterogeneous nuclear ribonuclear protein L-like (hnRNPLL) and reduced T cell function.

Heterogeneous nuclear ribonuclear protein l-like (hnRNPLL) is an RNA binding protein that regulates alternative splicing of mRNA and is abundantly expressed in memory T lymphocytes of the immune system and in the brain. A hypomorphic allele of the gene encoding hnRNPLL (Hnrpllthunder) selectively reduces T cell accumulation in lymphoid tissues, but little is known about its effects in the brain. Therefore, we exposed Hnrpllthunder mice to a test battery with relevance for a range of psychiatric illnesses. Thunder mice showed enhanced immobility in the tail-suspension test for depression-related behaviours, impaired short-term spatial memory in the Y-maze and reduced avoidance learning in the active avoidance test. Thus, in addition to its reported effects on immune function, the hnRNPLL mutation in thunder mice selectively affected aspects of behaviour.

The contribution of animal models to understanding the role of the immune system in human idiopathic pulmonary fibrosis.

Pulmonary fibrosis occurs in a heterogeneous group of lung disorders and is characterised by an excessive deposition of extracellular matrix proteins within the pulmonary interstitium, leading to impaired gas transfer and a loss of lung function. In the past 10 years, there has been a dramatic increase in our understanding of the immune system and how it contributes to fibrogenic processes within the lung. This review will compare some of the models used to investigate the pathogenesis and treatment of pulmonary fibrosis, in particular those used to study immune cell pathogenicity in idiopathic pulmonary fibrosis, highlighting their advantages and disadvantages in dissecting human disease.

Mesothelial cells regulate immune responses in health and disease: role for immunotherapy in malignant mesothelioma.

The mesothelium when first described was thought to function purely as a non-adhesive surface to facilitate intracoelomic movement of organs. However, the mesothelium is now recognized as a dynamic cellular membrane with many important functions that maintain serosal integrity and homeostasis. For example, mesothelial cells interact with and help regulate the body's inflammatory and immune system following infection, injury, or malignancy. With recent advances in our understanding of checkpoint molecules and the advent of novel immunotherapy approaches, there has been an increase in the number of studies examining mesothelial and immune cell interaction, in particular the role of these interactions in malignant mesothelioma. This review will highlight some of the recent advances in our understanding of how mesothelial cells help regulate serosal immunity and how in a malignant environment, the immune system is hijacked to stimulate tumor growth. Ways to treat mesothelioma using immunotherapy approaches will also be discussed.

Evolution and Adaptation of the Avian H7N9 Virus into the Human Host.

Influenza viruses arise from animal reservoirs, and have the potential to cause pandemics. In 2013, low pathogenic novel avian influenza A(H7N9) viruses emerged in China, resulting from the reassortment of avian-origin viruses. Following evolutionary changes, highly pathogenic strains of avian influenza A(H7N9) viruses emerged in late 2016. Changes in pathogenicity and virulence of H7N9 viruses have been linked to potential mutations in the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA), as well as the viral polymerase basic protein 2 (PB2). Recognizing that effective viral transmission of the influenza A virus (IAV) between humans requires efficient attachment to the upper respiratory tract and replication through the viral polymerase complex, experimental evidence demonstrates the potential H7N9 has for increased binding affinity and replication, following specific amino acid substitutions in HA and PB2. Additionally, the deletion of extended amino acid sequences in the NA stalk length was shown to produce a significant increase in pathogenicity in mice. Research shows that significant changes in transmissibility, pathogenicity and virulence are possible after one or a few amino acid substitutions. This review aims to summarise key findings from that research. To date, all strains of H7N9 viruses remain restricted to avian reservoirs, with no evidence of sustained human-to-human transmission, although mutations in specific viral proteins reveal the efficacy with which these viruses could evolve into a highly virulent and infectious, human-to-human transmitted virus.

Effect of exercise on acute postprandial glucose concentrations and interleukin-6 responses in sedentary and overweight males.

This study examined the effect of 2 forms of exercise on glucose tolerance and the concurrent changes in markers associated with the interleukin (IL)-6 pathways. Fifteen sedentary, overweight males (29.0 ± 3.1 kg/m2) completed 2 separate, 3-day trials in randomised and counterbalanced order. An oral glucose tolerance test (OGTT; 75 g) was performed at the same time on each day of the trial. Day 2 of each trial consisted of a single 30-min workload-matched bout of either high-intensity intermittent exercise (HIIE; alternating 100% and 50% of peak oxygen uptake) or continuous moderate-intensity exercise (CME; 60 % of peak oxygen uptake) completed 1 h prior to the OGTT. Venous blood samples were collected before, immediately after, 1 h after, and 25 h after exercise for measurement of insulin, C-peptide, IL-6, and the soluble IL-6 receptors (sIL-6R; soluble glycoprotein 130 (sgp130)). Glucose area under the curve (AUC) was calculated from capillary blood samples collected throughout the OGTT. Exercise resulted in a modest (4.4%; p = 0.003) decrease in the glucose AUC when compared with the pre-exercise AUC; however, no differences were observed between exercise conditions (p = 0.65). IL-6 was elevated immediately after and 1 h after exercise, whilst sgp130 and sIL-6R concentrations were reduced immediately after exercise. In summary, exercise was effective in reducing glucose AUC, which was attributed to improvements that took place between 60 and 120 min into the OGTT, and was in parallel with an increased ratio of IL-6 to sIL-6R, which accords with an increased activation via the "classical" IL-6 signalling pathway. Our findings suggest that acute HIIE did not improve glycaemic response when compared with CME.

Idiopathic pulmonary fibrosis and a role for autoimmunity.

Idiopathic pulmonary fibrosis (IPF) is the most common of the idiopathic interstitial pneumonias. It is typically associated with extensive and progressive fibrosis, and is fatal and has limited treatment options. Characteristically IPF patients display large lymphocyte aggregates composed of CD3+ T cells and CD20+ B cells within the lung tissue that are located near sites of active fibrosis. In addition, IPF patients can have autoantibodies to a range of host antigens, suggesting a breakdown in immunological tolerance. In this review, we examine the role of T and B cells in IPF pathogenesis and discuss how loss of self-tolerance to lung-specific proteins could exacerbate disease progression in IPF. We discuss what these results mean in terms of future prospects for immunotherapy of IPF.

The Role of Alternative Splicing in the Control of Immune Homeostasis and Cellular Differentiation.

Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system.

The role of ubiquitin ligases in the control of organ specific autoimmunity.

Diabetes mellitus is characterized by chronic hyperglycemia caused by a deficiency in insulin action, insulin secretion or both. Type 1 diabetes is classified as the destruction of beta cells leading to a deficiency in insulin production. Type1 diabetes accounts for 5-10% of patients with diabetes and most commonly is caused by the autoimmune destruction of the beta cells in the pancreas. The adaptive immune system is composed of antigen specific T and B lymphocytes which play a central role in protecting the human body from infectious pathogens but occasionally autoreactive T and B cells can escape immune tolerance, become activated and induce autoimmune diseases. Naïve T cells require two distinct signals one delivered via the antigen receptor and the second through the costimulatory receptor CD28 that leads to the induction of IL-2 gene transcription. IL-2 is an important T cell growth factor that can influence both immunity and tolerance. Given its pivotal role it is not surprising that the immune system places strict regulation over Il2 gene transcription that is controlled by a number of E3 ubiquitin ligases that modulate TCR and CD28 signaling. This review will examine how different E3 ligases function to control T effector cell differentiation and how studies in gene knockout animal models has been crucial in understanding how these proteins function in vivo to regulate immune tolerance in the peripheral circulation.

Differential requirement for the CD45 splicing regulator hnRNPLL for accumulation of NKT and conventional T cells.

Natural killer T (NKT) cells represent an important regulatory T cell subset that develops in the thymus and contains immature (NK1.1(lo)) and mature (NK1.1(hi)) cell subsets. Here we show in mice that an inherited mutation in heterogeneous ribonucleoprotein L-like protein (hnRNPLL(thunder)), that shortens the survival of conventional T cells, has no discernible effect on NKT cell development, homeostasis or effector function. Thus, Hnrpll deficiency effectively increases the NKT∶T cell ratio in the periphery. However, Hnrpll mutation disrupts CD45RA, RB and RC exon silencing of the Ptprc mRNA in both NKT and conventional T cells, and leads to a comparably dramatic shift to high molecular weight CD45 isoforms. In addition, Hnrpll mutation has a cell intrinsic effect on the expression of the developmentally regulated cell surface marker NK1.1 on NKT cells in the thymus and periphery but does not affect cell numbers. Therefore our results highlight both overlapping and divergent roles for hnRNPLL between conventional T cells and NKT cells. In both cell subsets it is required as a trans-acting factor to regulate alternative splicing of the Ptprc mRNA, but it is only required for survival of conventional T cells.

Cooperation between somatic Ikaros and Notch1 mutations at the inception of T-ALL.

To understand the interactions between Notch1 and Ikaros in the evolution of T cell acute lymphoblastic leukemia (T-ALL), we traced the evolution of T-ALL in mice with an inherited Ikaros mutation, Ikzf1(Plstc) which inactivates DNA binding. DNA-binding Ikaros repressed Notch1 response in transfected cell lines and in CD4(+)8(+) (DP) thymocytes from young pre-leukemic Ikzf1(Plstc) heterozygous mice. In DP thymocytes, a 50-1000 fold escalation in mRNA for Notch1 target genes Hes1 and Dtx1 preceded thymic lymphoma or leukemia and was closely correlated with the first detectable differentiation abnormalities, loss of heterozygosity (LOH) eliminating wild-type Ikzf1, and multiple missense and truncating Notch1 mutations. These findings illuminate the early stages of leukemogenesis by demonstrating progressive exaggeration of Notch1 responsiveness at the DP thymocyte stage brought about by multiple mutations acting in concert upon the Notch1 pathway.

Mechanisms that regulate peripheral immune responses to control organ-specific autoimmunity.

The immune system must balance the need to maintain a diverse repertoire of lymphocytes to be able to fight infection with the need to maintain tolerance to self-proteins. The immune system places strict regulation over the ability of T cells to produce the major T cell growth factor interleukin 2 as this cytokine can influence a variety of immune outcomes. T cells require the delivery of two signals, one through the antigen receptor and a second through the costimulatory receptor CD28. The immune system uses a variety of E3 ubiquitin ligases to target signaling proteins that function downstream of the TCR and CD28 receptors. Mutations in these E3 ligases can lead to a breakdown in immune tolerance and development of autoimmunity. This paper will examine the role of a range of E3 ubiquitin ligases and signaling pathways that influence the development of T-cell effector responses and the development of organ-specific autoimmune diseases such as type 1 diabetes.