Mucosa-associated lymphoid tissue (MALT) initiates immune responses at mucosal entry sites. Within MALT microfold (M) cells sample luminal antigens and deliver them to underlying immune cells. Despite their functional importance, few tools enable selective manipulation of M cells in vivo. Here we report the generation and characterization of a peptidoglycan recognition protein 1 (Pglyrp1-Cre knock-in mouse designed to allow conditional genetic access to M cells. Using Rosa26-tdTomato reporter mice, we found strong Pglyrp1 promoter activity in gut epithelial cells, including goblet and M cells, whereas activity in nasal-associated lymphoid tissue (NALT) was more heterogeneous and skewed towards immune cells, particularly neutrophils. To functionally interrogate Pglyrp1-expressing cells, we performed Cre-mediated ablation using three DTA-based models. The Rosa26GFP-DTA line caused marked perinatal lethality in double-positive pups, suggesting essential roles for Pglyrp1-positive cells early in life. In contrast, Rosa26DTA and Rosa26iDTR crosses produced minimal depletion of mucosal populations, including M cells, even at the highest non-lethal diphtheria toxin dose. These findings demonstrate tissue-specific Pglyrp1 promoter activity and highlight challenges in achieving M cell-specific targeting. Although not M cell-restricted, the Pglyrp1-Cre mouse provides a useful tool for manipulating Pglyrp1-expressing lineages and probing their roles in mucosal homeostasis and immunity.

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, yet the host pathways that regulate antigen presentation and lung inflammation during Mycobacterium tuberculosis (Mtb) infection are incompletely defined. Sorting nexin 5 (SNX5) is a protein implicated in endosomal trafficking, antigen processing, and antiviral host defense, but its contribution to antibacterial immunity is unknown. Here, we show that SNX5-deficient mice exhibit increased mortality following low-dose aerosol Mtb infection despite unchanged pulmonary bacterial burden compared with wild-type mice. Snx5-/- mice developed exacerbated lung inflammation without major alterations in immune cell recruitment. In macrophages, Snx5 did not affect phagocytosis, vacuolar maturation, intracellular bacterial control, or global transcriptional responses to Mtb but was required for efficient major histocompatibility complex (MHC) class II antigen presentation. Snx5 deficiency was associated with reduced endolysosomal proteolysis and impaired MHC class II antigen presentation in vitro, resulting in reduced activation of antigen-specific CD4+ T cells without altering surface MHC class II abundance or costimulatory molecule expression. Together, these findings identify SNX5 as a regulator of MHC class II antigen presentation that influences inflammatory outcomes during pulmonary Mtb infection.

Autophagy is a critical host defense mechanism that restricts intracellular pathogens such as Mycobacterium tuberculosis (Mtb). A key step in this process is the ubiquitination of Mtb or Mtb-associated structures. The E3 ligase SMURF1 catalyzes K48-linked ubiquitination, promoting bacterial clearance. However, the function of its homolog, SMURF2, in host defense remains undefined. Here, we demonstrate that Smurf2 deletion in murine macrophages increases SMURF1 levels, enhances LC3B lipidation, augments K48 ubiquitination of Mtb-associated structures, and reduces intracellular Mtb replication. These effects are reversed by Smurf1 deletion, supporting a role for SMURF1 in SMURF2-dependent control of Mtb. Mice with myeloid-specific Smurf2 deletion exhibit modestly prolonged survival following aerosol Mtb infection. In human macrophages, SMURF2 knockdown or its pharmacological inhibition with the HECT E3-ligase inhibitor Heclin reduces Mtb replication. Together, our findings identify SMURF2 as a negative regulator of macrophage control of Mtb and support further investigation of SMURF2 as a potential target for host-directed therapy in tuberculosis.

Tuberculosis (TB) spreads through the air, yet the physical conditions that permit effective airborne transmission remain poorly defined. Although early 20th-century guinea pig studies demonstrated efficient transmission of Mycobacterium tuberculosis (Mtb), this experimental paradigm has not been reestablished in modern high-containment facilities. Here, we show that airflow can impose constraints that suppress or permit biologically effective exposure between infected and susceptible hosts. Using a guinea pig model of animal-to-animal exposure, we combined transmission experiments with quantitative particle tracking and particle transport modeling to explain why some housing configurations fail to support effective exposure. Static environments and excessive unidirectional airflow prevented transmission, whereas controlled low-velocity airflow restored evidence of exposure, including tuberculin skin test conversion, antigen-specific immune responses, and pulmonary inflammation consistent with early infection. These findings identify airflow as a critical constraint on airborne TB transmission and establish a reproducible experimental framework for dissecting host, microbial, and environmental determinants of spread. IMPORTANCE:Tuberculosis remains one of the leading causes of death from infectious disease worldwide, and its spread depends on airborne transmission of Mycobacterium tuberculosis. Yet surprisingly little is known about the physical conditions that determine when transmission actually occurs. Classic experiments in guinea pigs demonstrated airborne spread of tuberculosis, but these systems have been difficult to reproduce in modern high-containment laboratories. In this study, we show that airflow conditions can determine whether pathogen-containing aerosols persist long enough to expose a susceptible host. By identifying airflow as a key constraint on transmission and establishing an experimental system that functions under contemporary biosafety conditions, this work provides a platform to experimentally dissect how microbial, host, and environmental factors interact to shape tuberculosis spread.

Host cell metabolic pathways influence innate immune responses to intracellular pathogens, but the contribution of nucleotide metabolism to antimicrobial defense remains incompletely defined. Here, we identify the mitochondrial nucleoside monophosphate kinase CMPK2 as a regulator of macrophage responses to Mycobacterium tuberculosis (Mtb). Using a targeted genetic screen of candidate host factors, we found that depletion of CMPK2 enhances intracellular Mtb replication in human macrophages. This phenotype was confirmed using both shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches. CMPK2 expression increased following macrophage activation and Mtb infection. Transcriptomic profiling revealed that loss of CMPK2 is associated with broad alterations in gene expression, including reduced expression of genes linked to innate immune and inflammatory responses early after infection. In contrast, myeloid-specific deletion of Cmpk2 in mice did not significantly alter bacterial burden or survival following aerosol Mtb infection, indicating that the contribution of CMPK2 to host defense is context dependent. Together, these findings identify CMPK2 as a host factor that limits Mtb replication in human macrophages and shapes innate immune gene expression programs.

Tuberculosis (TB) remains one of the leading causes of death from a single infectious agent worldwide, yet the host pathways that regulate antigen presentation and lung inflammation during Mycobacterium tuberculosis (Mtb) infection are incompletely defined. Sorting nexin 5 (SNX5) is a protein best known for roles in endosomal trafficking, antigen processing, and antiviral host defense, but its contribution to immunity during Mtb infection is unknown. Here, we show that SNX5-deficient mice exhibit markedly increased mortality following low-dose aerosol infection despite unchanged pulmonary bacterial burden compared to wild-type mice. Snx5 -/- mice developed exacerbated lung inflammation without major alterations in immune cell recruitment. In macrophages, Snx5 did not affect phagocytosis, vacuolar maturation, intracellular bacterial control, or global transcriptional responses to Mtb, but was required for efficient MHC class II antigen presentation. Snx5 deficiency reduced antigen degradation, limited peptide loading onto MHC-II and impaired activation of antigen-specific CD4+ T cells without altering surface MHC-II abundance or expression of costimulatory molecules. Together, these findings identify SNX5 as a previously unrecognized regulator of MHC-II peptide loading that shapes inflammatory outcomes during pulmonary Mtb infection, highlighting a role for the endosomal sorting machinery in immunity to intracellular pathogens.

Macroautophagy/autophagy enables macrophages to degrade intracellular Mycobacterium tuberculosis (Mtb), and this defense depends on E3 ubiquitin ligases such as PRKN/PARKIN/PARK2 and SMURF1, which tag Mtb-associated structures for lysosomal clearance. Deubiquitinases (DUBs) counter ubiquitin ligases by removing ubiquitin from molecular targets. We hypothesized that DUBs might offset ubiquitin ligase activity and negatively regulate host immunity to Mtb. Here, we identify USP15 (ubiquitin specific peptidase 15) as a negative regulator of MAP1LC3/LC3-dependent targeting pathways (consistent with xenophagy or CASM/LAP-related ATG8ylation) that mediate macrophage immunity to Mtb. Using a targeted knockdown screen in mouse macrophages, we found that Usp15 loss increased K63-linked ubiquitination and LC3 recruitment to Mtb-associated structures, leading to reduced bacterial replication. These effects required USP15's catalytic activity and were reversed by knockdown of PRKN or inhibition of autophagy initiation. In primary human macrophages, USP15 knockdown similarly enhanced LC3 targeting and restricted Mtb growth. Importantly, pharmacological inhibition of USP15 with a selective small molecule decreased Mtb burden in human macrophages. Our findings identify USP15 as a suppressor of macrophage immunity and suggest that targeting deubiquitinases may represent a promising host-directed therapeutic strategy against tuberculosis.Abbreviations: CFU: colony-forming unit; DUBs: deubiquitinases; K48-Ub: K48-linked ubiquitin; K63-Ub: K63-linked ubiquitin; Mtb-pLux: luminescent Mtb strain Mtb; Mycobacterium tuberculosis; MOI: multiplicity of infection; NTC: non-targeting control; TB: tuberculosis.

Innate immune cells, such as monocytes and macrophages, provide the earliest defense against intracellular pathogen infection by initiating signaling pathways and restricting pathogen replication. However, the full complement of proteins that mediate cell-autonomous immunity remains incompletely defined. Here, we applied cysteine-directed activity-based protein profiling (ABPP) to map proteome-wide cysteine reactivity changes in THP-1 monocytes and primary human monocyte-derived macrophages during Mycobacterium tuberculosis (Mtb) infection. Across both cell types, we quantified 148 cysteine residues with altered reactivity. Knockdown of a subset of proteins harboring infection-induced reactivity significantly altered Mtb replication in THP-1 monocytes, linking proteins with reactive cysteines to antimicrobial defense. These data define previously unrecognized host protein changes during Mtb infection and provide a resource for investigating post-translational events that regulate innate immune responses to intracellular bacteria.

Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. Although both two-dimensional cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human alveolar microenvironment. Here, we describe the development of a humanized, three-dimensional, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum lung infection that incorporates only primary human cells, such as pulmonary vascular endothelial cells, in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and in studies of new antibiotics.

Tuberculosis (TB) is transmitted through the air, yet the determinants of natural airborne transmission remain poorly defined. Early twentieth-century guinea pig studies demonstrated efficient airborne transmission of Mycobacterium tuberculosis (Mtb), but this paradigm has not been reestablished in contemporary containment facilities. Here, we show that ventilation can impose airflow constraints that suppress transmission under otherwise permissive conditions. Using a guinea pig model of animal-to-animal exposure, we combined transmission experiments with quantitative airflow measurements and particle transport modeling to explain why some housing configurations fail to support effective exposure. Static environments and excessive unidirectional airflow prevented transmission, whereas controlled low-velocity airflow restored evidence of exposure, including tuberculin skin test conversion, antigen-specific immune responses, and pulmonary inflammation consistent with early infection. These findings identify airflow as a critical constraint on airborne TB transmission and provide a reproducible framework to dissect host, microbial, and environmental determinants of spread.

Autophagy enables macrophages to degrade intracellular Mycobacterium tuberculosis (Mtb), and this defense depends on E3 ubiquitin ligases such as PARKIN and SMURF1, which tag Mtb-associated structures for lysosomal clearance. Deubiquitinases (DUBs) counter ubiquitin ligases by removing ubiquitin from molecular targets. We hypothesized that DUBs might offset ubiquitin ligase activity and negatively regulate host immunity to Mtb. Here, we identify ubiquitin-specific protease 15 (USP15) as a negative regulator of autophagy-mediated macrophage immunity to Mtb. Using a targeted knockdown screen in mouse macrophages, we found that Usp15 loss increased K63-linked ubiquitination and LC3 recruitment to Mtb-associated structures, leading to reduced bacterial replication. These effects required USP15’s catalytic activity and were reversed by knockdown of PARKIN ( Park2 ) or inhibition of autophagy initiation. In primary human macrophages, USP15 knockdown similarly enhanced LC3 targeting and restricted Mtb growth. Importantly, pharmacologic inhibition of USP15 with a selective small molecule decreased Mtb burden in human macrophages. Our findings identify USP15 as a suppressor of macrophage immunity and suggest that targeting deubiquitinases may represent a promising host-directed therapeutic strategy against tuberculosis. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69, U19 AI142784 National Heart Lung and Blood Institute, T32HL098040 Office of the Director, 1S10OD028630

The nasal, oropharyngeal, and bronchial mucosa are primary contact points for airborne pathogens like Mycobacterium tuberculosis (Mtb), SARS-CoV-2, and influenza virus. While mucosal surfaces can function as both entry points and barriers to infection, mucosa-associated lymphoid tissues (MALT) facilitate early immune responses to mucosal antigens. MALT contains a variety of specialized epithelial cells, including a rare cell type called a microfold cell (M cell) that functions to transport apical antigens to basolateral antigen-presenting cells, a crucial step in the initiation of mucosal immunity. M cells have been extensively characterized in the gastrointestinal (GI) tract in murine and human models. However, the precise development and functions of human airway M cells are unknown. Here, using single-nucleus RNA sequencing (snRNA-seq), we generated an atlas of cells from the human adenoid and identified 26 unique cell types representing basal, club, hillock, and hematopoietic lineages, defined their developmental trajectories, and determined cell-cell relationships. Using trajectory analysis, we found that human airway M cells develop from progenitor club cells and express a gene signature distinct from intestinal M cells. Surprisingly, we also identified a heretofore unknown epithelial cell type demonstrating a robust interferon-stimulated gene signature. Our analysis of human adenoid cells enhances our understanding of mucosal immune responses and the role of M cells in airway immunity. This work also provides a resource for understanding early interactions of pathogens with airway mucosa and a platform for development of mucosal vaccines.

Cough is a hallmark sign of tuberculosis and a key driver of transmission. Although traditionally attributed to host-driven inflammation, we previously demonstrated that Mycobacterium tuberculosis lipid extract (Mtb extract) and its component sulfolipid-1 (SL-1) directly act on nociceptive neurons to induce cough in guinea pigs. However, the cellular mechanisms by which Mtb extract and SL-1 modulate nociceptive sensory neurons remain incompletely understood. Using calcium imaging, we found that Mtb extract and SL-1 increased intracellular Ca2+ signals in TRPV1+ neurons from both mouse nodose and human dorsal root ganglia (hDRG). We observed that YM254890 (a Gαq/11 inhibitor) could attenuate these Ca2+ signaling events, even in the absence of extracellular Ca2+, suggesting a G protein-coupled receptor (GPCR)-mediated mechanism driven by Gαq/11 signaling to intracellular Ca2+ stores. Mtb extract treatment also enhanced action potential (AP) generation in mouse nodose nociceptors via an SL-1-dependent mechanism. Mtb extract increased the number and half-width of evoked APs, indicating direct modulation of voltage-gated ion channel activity. The Mtb extract-induced change in mouse nodose neuron excitability and in the AP half-width was blocked by YM254890 treatment. Taken together, these findings link TB pathogen-derived lipids to GPCR signaling that directly increases the excitability of sensory neurons.NEW & NOTEWORTHY Cough elicited by TB facilitates disease transmission; however, the underlying neuronal mechanisms responsible for this phenomenon are unknown. Our study demonstrates that Mtb lipid sulpholipid-1 can activate sensory neurons directly through Gαq/11-mediated mobilization of intracellular calcium stores and enhance neuronal excitability. These effects can be blocked by YM254890. These findings reveal a GPCR-mediated mechanism linking bacterial virulence to changes in neuronal excitability, identifying potential therapeutic targets for treating cough associated with TB.

Cough drives respiratory pathogen transmission, yet how microbes directly engage host sensory neurons to trigger cough is largely unknown. We previously demonstrated that the Mycobacterium tuberculosis (Mtb) glycolipid sulfolipid-1 (SL-1) activates neurons and induces cough. Here, we reveal that phenolic glycolipid (PGL) produced by the hypertransmissible HN878 Mtb strain activates both mouse and human nociceptive neurons in vitro using calcium imaging and electrophysiology and is sufficient to induce cough using plethysmography. Combined with SL-1, PGL potently triggers neuronal activation. By synthesizing various PGL analogs, we show that neuroactivity is proportional to saccharide chain length and structure. Mechanistically, PGL stimulates rapid extracellular ATP release, which engages neuronal P2X3 purinergic receptors-an effect blocked by a P2X3 antagonist. These findings uncover a neuronal activation pathway co-opted by certain Mtb strains to enhance transmission via cough and suggest inhibition of purinergic signaling as a potential strategy to block airborne spread of Mtb.

Tuberculosis (TB) spreads through the air when Mycobacterium tuberculosis (Mtb) passes from infected to susceptible hosts, yet the environmental, biophysical and microbial factors governing this process remain poorly understood. In the early twentieth century, Perla and Lurie used guinea pigs to demonstrate natural airborne transmission of Mtb, but such studies have not been revisited in the modern biosafety era. Here, we developed an aerodynamically-optimized guinea pig housing system that models natural, airborne, animal-to-animal Mtb transmission under biosafety level 3 (BSL-3) containment. Iterative engineering and particle transport experiments revealed that airflow is a critical determinant of transmission efficiency. Static housing and excessive unidirectional ventilation both eliminated transmission, whereas controlled, low-velocity airflow enabled aerosol particle retention and exposure of naïve animals. Under these optimized conditions, recipient guinea pigs converted their tuberculin skin tests above a defined positive threshold, developed Mtb-specific antibody responses, and exhibited pulmonary inflammation consistent with infection. These findings demonstrate that flow rates govern natural transmission of Mtb and provide a reproducible small-animal model for studying bacterial, host, and environmental factors that drive infectious spread. By reviving a century-old experimental paradigm with modern physics, engineering and immunologic tools, this work establishes a platform to dissect the mechanisms underlying airborne transmission of tuberculosis.

Cough is a hallmark sign of tuberculosis and key driver of transmission. While traditionally attributed to host-driven inflammation, we previously demonstrated that Mycobacterium tuberculosis lipid extract (Mtb extract) and its component sulfolipid-1 (SL-1) directly activate nociceptive neurons to induce cough in guinea pigs. However, the cellular mechanisms by which Mtb extract and SL-1 modulate nociceptive sensory neurons remain incompletely understood. Here, we show that Mtb extract enhances action potential (AP) generation in mouse nodose nociceptors via an SL-1-dependent mechanism. Using calcium imaging, we found that Mtb extract and SL-1 increased intracellular Ca2+ signals in TRPV1+ neurons from both mouse nodose and human dorsal root ganglia (hDRG). These Ca2+ signals were attenuated by the Gαq/11 pathway inhibitor YM254890, even in the absence of extracellular Ca2+, suggesting involvement of intracellular Ca2+ stores. Together, these findings indicate that SL-1 engages Gαq/11-coupled pathways to sensitize nociceptors via intracellular Ca2+ release, providing mechanistic insight into tuberculosis-associated cough and potential targets for therapeutic intervention.

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss-of-function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found that deletion of CDKL5 or expression of a clinically relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. Cdkl5-knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.

Surface lipids on pathogenic mycobacteria modulate infection outcomes by regulating host immune responses. Phenolic glycolipid (PGL) is a host-modulating surface lipid that varies among clinical Mycobacterium tuberculosis strains. PGL is also found in Mycobacterium marinum, where it promotes infection of zebrafish through effects on the innate immune system. Given the important role this lipid plays in the host-pathogen relationship, tools for profiling its abundance, spatial distribution, and dynamics are needed. Here, we report a strategy for imaging PGL in live mycobacteria using bioorthogonal metabolic labeling. We functionalized the PGL precursor p-hydroxybenzoic acid (pHB) with an azide group (3-azido pHB). When fed to mycobacteria, 3-azido pHB was incorporated into the cell surface, which could then be visualized via the bioorthogonal conjugation of a fluorescent probe. We confirmed that 3-azido pHB incorporates into PGL using mass spectrometry methods and demonstrated selectivity for PGL-producing M. marinum and M. tuberculosis strains. Finally, we applied this metabolic labeling strategy to study the dynamics of PGL within the mycobacterial membrane. This new tool enables visualization of PGL that may facilitate studies of mycobacterial pathogenesis.

Coughing is a dynamic physiological process resulting from input of vagal sensory neurons innervating the airways and perceived airway irritation. Although cough serves to protect and clear the airways, it can also be exploited by respiratory pathogens to facilitate disease transmission. Microbial components or infection-induced inflammatory mediators can directly interact with sensory nerve receptors to induce a cough response. Analysis of cough generated aerosols and transmission studies have further demonstrated how infectious disease is spread through coughing. This review summarizes the neurophysiology of cough, cough induction by respiratory pathogens and inflammation, and cough-mediated disease transmission.