A narrative review of the controversy on the risk of mycobacterial infections with immune checkpoint inhibitor use: does Goldilocks have the answer?

Background and Objective: Immune checkpoint inhibitors (ICIs) have revolutionized oncologic treatment. Whether ICIs increase susceptibility to or provide protection against mycobacterial infections remains controversial. The objective of this narrative review is to summarize the literature on the link between ICI use and mycobacterial infections-tuberculosis and non-tuberculous mycobacterial (NTM) infections-and to critically discuss evidence linking ICIs with mycobacterial infections, the possible confounders, and, if indeed the ICIs predispose to such infections, the potential mechanisms of how this may occur. Methods: We conducted a literature search on PubMed for relevant articles published from 2011 to current time [2024] utilizing specific keywords of "immune checkpoint inhibitors", "programmed cell death protein-1", "PD-1", "programmed death-ligand 1", "PD-L1", "cytotoxic T-lymphocyte-associated protein-4", or "CTLA-4" with that of "non-tuberculous mycobacterial lung disease", "tuberculosis", or "mycobacteria". The bibliographies of identified papers were perused for additional relevant articles. Key Content and Findings: Ex vivo studies using human cells indicate that ICIs would be salubrious for the host against mycobacteria. Yet, many case reports associate ICI use with mycobacterial infections, mostly tuberculosis. Potential confounders include immunosuppression from the cancer, concomitant use of immunosuppressive drugs, lung injury and distortion from chemotherapeutics or radiation, and reporting bias. Mice with genetic disruption of the programmed cell death protein-1 (PD-1) gene are paradoxically more susceptible to Mycobacterium tuberculosis (M. tuberculosis). In contrast, mice administered neutralizing antibody to T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) or knocked out for TIM3 gene have greater capacity to control an M. tuberculosis infection. We posit that hosts with greater baseline immunodeficiency are more likely to derive benefit from ICIs against mycobacterial infections than those with more intact immunity, where ICIs are more likely to be detrimental. Conclusions: Studies are needed to test the hypothesis that ICIs may either protect or predispose to mycobacterial infections, depending on the baseline host immune status. Prospective studies are required of patients on ICIs that control for potential confounders as anecdotal case reports are insufficient to provide a causal link. Murine studies with ICIs are also required to corroborate or refute studies of mice with genetic disruption of an immune checkpoint.

Changes in gut microbiome correlate with intestinal barrier dysfunction and inflammation following a 3-day ethanol exposure in aged mice.

Alcohol use among older adults is on the rise. This increase is clinically relevant as older adults are at risk for increased morbidity and mortality from many alcohol-related chronic diseases compared to younger patients. However, little is known regarding the synergistic effects of alcohol and age. There are intriguing data suggesting that aging may lead to impaired intestinal barrier integrity and dysbiosis of the intestinal microbiome, which could increase susceptibility to alcohol's negative effects. To study the effects of alcohol in age we exposed aged and young mice to 3 days of moderate ethanol and evaluated changes in gut parameters. We found that these levels of drinking do not have obvious effects in young mice but cause significant alcohol-induced gut barrier dysfunction and expression of the pro-inflammatory cytokine TNFα in aged mice. Ethanol-induced downregulation of expression of the gut-protective antimicrobial peptides Defa-rs1, Reg3b, and Reg3g was observed in aged, but not young mice. Analysis of the fecal microbiome revealed age-associated shifts in microbial taxa, which correlated with intestinal and hepatic inflammatory gene expression. Taken together, these data demonstrate that age drives microbiome dysbiosis, while ethanol exposure in aged mice induces changes in the expression of antimicrobial genes important for separating these potentially damaging microbes from the intestinal lumen. These changes highlight potential mechanistic targets for prevention of the age-related exacerbation of effects of ethanol on the gut.

Advanced age is associated with changes in alveolar macrophages and their responses to the stress of traumatic injury.

Alveolar macrophages (AMs) are tissue-resident cells of the lower airways that perform many homeostatic functions critical for pulmonary health and protection against pathogens. However, little is known about the factors that shape AMs during healthy aging. In these studies, we sought to characterize age-related changes in AM phenotype, function, and responses to a physiologic stressor, that is, distal injury. Age was associated with a wide range of changes in cell surface receptor and gene expression by AMs, reflecting a unique alternatively activated phenotype. AMs from aged mice also exhibited markers of cellular senescence along with down-regulation of genes involved in growth and cell cycle pathways relative to young controls. Furthermore, AMs from aged mice showed a stunted transcriptional response to distal injury compared with AMs from young mice. Many changes were found to involve glucocorticoid-regulated genes, and corticosteroid treatment of primary AMs ex vivo revealed diminished transcriptional responses in cells from aged animals. These results demonstrate that there is a complex age-dependent AM phenotype associated with dysregulated stress hormone signaling that may interfere with AM responses to physiologic stressors and could contribute to AM dysfunction and the decline of pulmonary immunity during healthy aging.

Ethanol Intoxication Impairs Respiratory Function and Bacterial Clearance and Is Associated With Neutrophil Accumulation in the Lung After Streptococcus pneumoniae Infection.

Alcohol consumption is commonplace in the United States and its prevalence has increased in recent years. Excessive alcohol use is linked to an increased risk of infections including pneumococcal pneumonia, mostly commonly caused by Streptococcus pneumoniae. In addition, pneumonia patients with prior alcohol use often require more intensive treatment and longer hospital stays due to complications of infection. The initial respiratory tract immune response to S. pneumoniae includes the production of pro-inflammatory cytokines and chemokines by resident cells in the upper and lower airways which activate and recruit leukocytes to the site of infection. However, this inflammation must be tightly regulated to avoid accumulation of toxic by-products and subsequent tissue damage. A majority of previous work on alcohol and pneumonia involve animal models utilizing high concentrations of ethanol or chronic exposure and offer conflicting results about how ethanol alters immunity to pathogens. Further, animal models often employ a high bacterial inoculum which may overwhelm the immune system and obscure results, limiting their applicability to the course of human infection. Here, we sought to determine how a more moderate ethanol exposure paradigm affects respiratory function and innate immunity in mice after intranasal infection with 104 colony forming units of S. pneumoniae. Ethanol-exposed mice displayed respiratory dysfunction and impaired bacterial clearance after infection compared to their vehicle-exposed counterparts. This altered response was associated with increased gene expression of neutrophil chemokines Cxcl1 and Cxcl2 in whole lung homogenates, elevated concentrations of circulating granulocyte-colony stimulating factor (G-CSF), and higher neutrophil numbers in the lung 24 hours after infection. Taken together, these findings suggest that even a more moderate ethanol consumption pattern can dramatically modulate the innate immune response to S. pneumoniae after only 3 days of ethanol exposure and provide insight into possible mechanisms related to the compromised respiratory immunity seen in alcohol consumers with pneumonia.

Age-Related Intestinal Dysbiosis and Enrichment of Gut-specific Bacteria in the Lung Are Associated With Increased Susceptibility to Streptococcus pneumoniae Infection in Mice.

The portion of the global population that is over the age of 65 is growing rapidly and this presents a number of clinical complications, as the aged population is at higher risk for various diseases, including infection. For example, advanced age is a risk factor for heightened morbidity and mortality following infection with Streptococcus pneumoniae. This increased vulnerability is due, at least in part, to age-related dysregulation of the immune response, a phenomenon termed immunosenescence. However, our understanding of the mechanisms influencing the immunosenescent state and its effects on the innate immune response to pneumonia remain incomplete. Recently, a role for the gut microbiome in age-specific alterations in immunity has been described. Here, we utilized a murine model of intranasal Streptococcus pneumoniae infection to investigate the effects of age on both the innate immune response and the intestinal microbial populations after infection. In aged mice, compared to their younger counterparts, infection with Streptococcus pneumoniae led to increased mortality, impaired lung function and inadequate bacterial control. This poor response to infection was associated with increased influx of neutrophils into the lungs of aged mice 24 h after infection. The exacerbated pulmonary immune response was not associated with increased pro-inflammatory cytokines in the lung compared to young mice but instead heightened expression of immune cell recruiting chemokines by lung neutrophils. Bacterial 16S-rRNA gene sequencing of the fecal microbiome of aged and young-infected mice revealed expansion of Enterobacteriaceae in the feces of aged, but not young mice, after infection. We also saw elevated levels of gut-derived bacteria in the lung of aged-infected mice, including the potentially pathogenic symbiote Escherichia coli. Taken together, these results reveal that, when compared to young mice, Streptococcus pneumoniae infection in age leads to increased lung neutrophilia along with potentially pathogenic alterations in commensal bacteria and highlight potential mechanistic targets contributing to the increased morbidity and mortality observed in infections in age.

Advanced age exacerbates intestinal epithelial permeability after burn injury in mice.

BACKGROUND: Advanced age is an independent risk factor for morbidity and mortality after burn injury. Following burn, the intestines can become permeable leading to the leakage of bacteria and their products from the lumen of the ileum to the portal and systemic circulation. Here, we sought to determine the effects of advanced age on intestinal permeability post burn injury and assess intestinal inflammatory biomarkers. METHODS: Young (4-5 months) and aged (18-22 months) female BALB/cBy mice were subjected to a 12-15% total body surface area (TBSA) sham or burn injury. 24 h after injury, mice were euthanized, and organs collected. Colony-forming units (CFU) were counted from plated mesenteric lymph nodes (MLN). Gene expression of ileal tight junctional proteins, occludin and zonula occludens 1 (ZO-1), in addition to ileal damage associated molecular pattern (DAMP) proteins, S100A8 and S100A9, as well as ileal inflammatory markers IL-6 and TNF-α were measured by qPCR. Intestinal cell death was measured by ELISA. Intestinal permeability was determined by FITC fluorescence in serum; 4kD FITC-dextran was given by oral gavage 3 h before euthanasia. RESULTS: Aged mice subjected to burn injury had increased intestinal permeability as evidenced by a 5.8-fold higher level of FITC-dextran in their serum when compared to all other groups (p < 0.05). In addition, aged burn-injured mice exhibited heightened bacterial accumulation in the MLN with a 15.5-fold increase over all other groups (p < 0.05). Histology of ileum failed to show differences in villus length among all groups. Analysis of ileal tight junctional proteins and inflammatory marker gene expression revealed no difference in Ocln, Tjp1, Il6, or Tnf expression among all groups, but 2.3 and 2.9-fold upregulation of S100a8 and S100a9, respectively, in aged burn-injured mice relative to both young groups and aged sham-injured mice (p < 0.05). Lastly, cell death in the ileum was elevated more than two-fold in aged burn-injured mice relative to young animals regardless of injury (p < 0.05). CONCLUSIONS: These data demonstrate that advanced age exacerbates intestinal epithelial permeability after burn injury. Heightened apoptosis may be responsible for the elevated intestinal leakiness and accumulation of bacteria in mesenteric lymph nodes. In addition, S100a8/9 may serve as a biomarker of elevated inflammation within the intestine.

A novel murine model of multi-day moderate ethanol exposure reveals increased intestinal dysfunction and liver inflammation with age.

BACKGROUND: There are currently > 600 million people over the age of 65 globally and this number is expected to double by the year 2050. Alcohol use among this population is on the rise, which is concerning as aging is associated with increased risk for a number of chronic illnesses. As most studies investigating the effects of alcohol have focused on young/middle-aged populations, there is a dearth of information regarding the consequences of alcohol use in older consumers. In addition, most murine ethanol models have concentrated on exposure to very high levels of ethanol, while the vast majority of elderly drinkers do not consume alcohol in excess; instead, they drink on average 2 alcoholic beverages a day, 3-4 days a week. METHODS: We designed a murine model of aging and moderate ethanol consumption to determine if the deleterious effects of alcohol on the gut-liver axis are exacerbated in aged, relative to younger, animals. Aged and young mice were exposed to a multi-day moderate exposure ethanol regimen for 4 weeks and changes in gut permeability along with intestinal tight junction protein and antimicrobial peptide gene expression were measured. In addition, hepatic inflammation was assessed by histological analysis, inflammatory gene expression and flow cytometric analysis of inflammatory infiltrate. RESULTS: Our results reveal that in aged, but not young mice, moderate ethanol exposure yielded significantly worsened intestinal permeability, including increased bacterial translocation from the gut, elevated serum iFABP and leakage of FITC-dextran from the gut. Interestingly, moderate ethanol exposure in young animals led to gut protective transcriptional changes in the ileum while this protective response was blunted in aged mice. Finally, moderate ethanol exposure in aged mice also resulted in marked inflammatory changes in the liver. CONCLUSIONS: These results demonstrate that aged mice are more susceptible to ethanol-induced gut barrier dysfunction and liver inflammation, even at moderate doses of ethanol. This increased vulnerability to ethanol's gastrointestinal effects has important implications for alcohol use in the aging population. Future studies will explore whether improving intestinal barrier function can reverse these age-related changes.

Age-related changes in intestinal immunity and the microbiome.

The gastrointestinal (GI) tract is a vitally important site for the adsorption of nutrients as well as the education of immune cells. Homeostasis of the gut is maintained by the interplay of the intestinal epithelium, immune cells, luminal Ags, and the intestinal microbiota. The well-being of the gut is intrinsically linked to the overall health of the host, and perturbations to this homeostasis can have severe impacts on local and systemic health. One factor that causes disruptions in gut homeostasis is age, and recent research has elucidated how critical systems within the gut are altered during the aging process. Intestinal stem cell proliferation, epithelial barrier function, the gut microbiota, and the composition of innate and adaptive immune responses are all altered in advanced age. The aging population continues to expand worldwide, a phenomenon referred to as the "Silver Tsunami," and every effort must be made to understand how best to prevent and treat age-related maladies. Here, recent research about changes observed in the intestinal epithelium, the intestinal immune system, the microbiota, and how the aging gut interacts with and influences other organs such as the liver, lung, and brain are reviewed. Better understanding of these age-related changes and their impact on multi-organ interactions will aid the development of therapies to increase the quality of life for all aged individuals.

Advanced Age Impairs Intestinal Antimicrobial Peptide Response and Worsens Fecal Microbiome Dysbiosis Following Burn Injury in Mice.

Maintenance of the commensal bacteria that comprise the gut microbiome is essential to both gut and systemic health. Traumatic injury, such as burn, elicits a number of changes in the gut, including a shift in the composition of the microbiome (dysbiosis), increased gut leakiness, and bacterial translocation into the lymphatic system and bloodstream. These effects are believed to contribute to devastating secondary complications following burn, including pneumonia, acute respiratory distress syndrome, multi-organ failure, and septic shock. Clinical studies demonstrate that advanced age causes a significant increase in mortality following burn, but the role of the gut in this age-dependent susceptibility has not been investigated. In this study, we combined our well-established murine model of scald burn injury with bacterial 16S-rRNA gene sequencing to investigate how burn injury affects the fecal microbiome in aged versus young mice. Of our treatment groups, the most substantial shift in gut microbial populations was observed in aged mice that underwent burn injury. We then profiled antimicrobial peptides (AMPs) in the ileum, and found that burn injury stimulated a 20-fold rise in levels of regenerating islet-derived protein 3 gamma (Reg3γ), a 16-fold rise in regenerating islet-derived protein 3 beta (Reg3ß), and an 8-fold rise in Cathelicidin-related antimicrobial peptide (Cramp) in young, but not aged mice. Advanced age alone elicited 5-fold higher levels of alpha defensin-related sequence1 (Defa-rs1) in the ileum, but this increase was lost following burn. Comparison of bacterial genera abundance and AMP expression across treatment groups revealed distinct correlation patterns between AMPs and individual genera. Our results reveal that burn injury drives microbiome dysbiosis and altered AMP expression in an age-dependent fashion, and highlight potential mechanistic targets contributing to the increased morbidity and mortality observed in elderly burn patients.

Summary of the 2017 Alcohol and Immunology Research Interest Group (AIRIG) meeting.

On June 24, 2017, the 22nd annual Alcohol and Immunology Research Interest Group (AIRIG) meeting was held as a satellite conference during the annual Research Society on Alcoholism (RSA) Scientific Meeting in Denver, Colorado. The 2017 meeting focused broadly on mechanisms that link alcohol to tissue injury and inflammation, and how this research can be translated to improve human health. Two plenary sessions composed the meeting, which first explored the association between alcohol and trauma/tissue injury, and finished with a discussion of alcohol and mucosal inflammation. The presentations encompassed diverse areas of alcohol research, from effects on the brain, to airway and pulmonary systems, to gut barrier disruption. The discussions also thoughtfully highlighted how current laboratory and clinical research can be used to prevent or treat alcohol-related morbidity and mortality.

Complement Component C1q Programs a Pro-Efferocytic Phenotype while Limiting TNFα Production in Primary Mouse and Human Macrophages.

Deficiency in complement component C1q is associated with an inability to clear apoptotic cells (efferocytosis) and aberrant inflammation in lupus, and identification of the pathways involved in these processes should reveal important regulatory mechanisms in lupus and other autoimmune or inflammatory diseases. In this study, C1q-dependent regulation of TNFα/IL-6 expression and efferocytosis was investigated using primary mouse bone marrow-derived macrophages and human monocyte-derived macrophages. C1q downregulated LPS-dependent TNFα production in mouse and human macrophages. While prolonged stimulation with C1q (18 h) was required to elicit a dampening of TNFα production from mouse macrophages, the human macrophages responded to C1q with immediate downregulation of TNFα. IL-6 production was unchanged in mouse and upregulated by human macrophages following prolonged stimulation with C1q. Our previous studies indicated that C1q programmed enhanced efferocytosis in mouse macrophages by enhancing expression of Mer tyrosine kinase and its ligand Gas6, a receptor-ligand pair that also inhibits proinflammatory signaling. Here, we demonstrated that C1q-dependent programming of human macrophage efferocytosis required protein synthesis; however, neither Mer nor the related receptor Axl was upregulated in human cells. In addition, while the C1q-collagen-like tails are sufficient for promoting C1q-dependent phagocytosis of antibody-coated targets, the C1q-tails failed to program enhanced efferocytosis or dampen TNFα production. These data further elucidate the mechanisms by which C1q regulates proinflammatory signaling and efferocytosis in macrophages, functions that are likely to influence the progression of autoimmunity and chronic inflammation.

Multiple Plasmids Contribute to Antibiotic Resistance and Macrophage Survival In Vitro in CMY2-Bearing Salmonella enterica.

Multiple drug resistance (MDR) in bacteria represents a notable problem but if carried on plasmid their spread could become a significant threat to public health. Plasmids in members of the Enterobacteriaceae family and in particular Salmonella and Escherichia coli strains have been implicated in the spread of antibiotic resistance genes. However, the mechanisms involved in the transfer of plasmid-borne resistance genes are not fully understood. Here, we analyzed the ability of Salmonella enterica clinical isolates to transfer plasmid-borne MDR to E. coli. We also determined whether possession of an Inc A/C plasmid by a S. enterica isolate would confer increased fitness compared to an isolate not carrying the plasmid. Sixteen human and animal isolates of S. enterica were screened using a three-panel multiplex PCR assay, and simplex PCR for the blaCMY-2 gene. Using these data we selected a suitable strain as a plasmid donor for the construction of a new Salmonella strain with an Inc A/C plasmid. This allowed us to compare isogenic strains with and without the Inc A/C plasmid in multiple growth, fitness, and invasion assays. The results showed that possession of Inc A/C plasmid confers significant fitness advantage when tested in J774 macrophages as opposed to HEp-2 cells where no significant difference was found. In addition, stress assays performed in vitro showed that the possession of this large plasmid by Salmonella strains tested here does not appear to incur a significant fitness cost. Gaining a better understanding of molecular mechanisms of plasmid transfer between pathogenic bacteria will allow us to characterize the role of MDR in pathogenicity of bacteria and to identify methods to reduce the frequency of dissemination of multiple antibiotic resistance genes.

Increased resistance to multiple antimicrobials and altered resistance gene expression in CMY-2-positive Salmonella enterica following a simulated patient treatment with ceftriaxone.

Salmonellosis is one of the most common causes of food-borne disease in the United States. Increasing antimicrobial resistance and corresponding increases in virulence present serious challenges. Currently, empirical therapy for invasive Salmonella enterica infection includes either ceftriaxone or ciprofloxacin (E. L. Hohmann, Clin. Infect. Dis. 32:263-269, 2001). The bla(CMY-2) gene confers resistance to ceftriaxone, the antimicrobial of choice for pediatric patients with invasive Salmonella enterica infections, making these infections especially dangerous (J. M. Whichard et al., Emerg. Infect. Dis. 11:1464-1466, 2005). We hypothesized that bla(CMY-2)-positive Salmonella enterica would exhibit increased MICs to multiple antimicrobial agents and increased resistance gene expression following exposure to ceftriaxone using a protocol that simulated a patient treatment in vitro. Seven Salmonella enterica strains survived a simulated patient treatment in vitro and, following treatment, exhibited a significantly increased ceftriaxone MIC. Not only would these isolates be less responsive to further ceftriaxone treatment, but because the bla(CMY-2) genes are commonly located on large, multidrug-resistant plasmids, increased expression of the bla(CMY-2) gene may be associated with increased expression of other drug resistance genes located on the plasmid (N. D. Hanson and C. C. Sanders, Curr. Pharm. Des. 5:881-894, 1999). The results of this study demonstrate that a simulated patient treatment with ceftriaxone can alter the expression of antimicrobial resistance genes, including bla(CMY-2) and floR in S. enterica serovar Typhimurium and S. enterica serovar Newport. Additionally, we have shown increased MICs following a simulated patient treatment with ceftriaxone for tetracycline, amikacin, ceftriaxone, and cefepime, all of which have resistance genes commonly located on CMY-2 plasmids. The increases in resistance observed are significant and may have a negative impact on both public health and antimicrobial resistance of Salmonella enterica.