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Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1β. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response. Modlin and colleagues examined the skin lesions of human leprosy patients using single-cell RNA sequencing coupled to cellular spatial mapping. Their analysis maps the architecture of granulomas in leprosy lesions from patients with leprosy with localized disease (tuberculoid leprosy, reversal reaction) to those with progressive infection (lepromatous leprosy).

TH17 cell subpopulations have been defined that contribute to inflammation and homeostasis, yet the characteristics of TH17 cells that contribute to host defense against infection are not clear. To elucidate the antimicrobial machinery of the TH17 subset, we studied the response to Cutibacterium acnes, a skin commensal that is resistant to IL-26, the only known TH17-secreted protein with direct antimicrobial activity. We generated C. acnes-specific antimicrobial TH17 clones (AMTH17) with varying antimicrobial activity against C. acnes, which we correlated by RNA sequencing to the expression of transcripts encoding proteins that contribute to antimicrobial activity. Additionally, we validated that AMTH17-mediated killing of C. acnes and bacterial pathogens was dependent on the secretion of granulysin, granzyme B, perforin, and histone H2B. We found that AMTH17 cells can release fibrous structures composed of DNA decorated with histone H2B that entangle C. acnes that we call T cell extracellular traps (TETs). Within acne lesions, H2B and IL-17 colocalized in CD4+ T cells, in proximity to TETs in the extracellular space composed of DNA decorated with H2B. This study identifies a functionally distinct subpopulation of TH17 cells with an ability to form TETs containing secreted antimicrobial proteins that capture and kill bacteria.

The initial interaction between a microbial pathogen and the host immune response influences the outcome of the battle between the host and the foreign invader. Leprosy, caused by the obligate intracellular pathogen Mycobacterium leprae, provides a model to study relevant human immune responses. Previous studies have adopted a targeted approach to investigate host response to M. leprae infection, focusing on the induction of specific molecules and pathways. By measuring the host transcriptome triggered by M. leprae infection of human macrophages, we were able to detect a host gene signature 24-48 hours after infection characterized by specific innate immune pathways involving the cell fate mechanisms autophagy and apoptosis. The top upstream regulator in the M. leprae-induced gene signature was NUPR1, which is found in the M. leprae-induced cell fate pathways. The induction of NUPR1 by M. leprae was dependent on the production of the type I interferon (IFN), IFN-β. Furthermore, NUPR1 mRNA and protein were upregulated in the skin lesions from patients with the multibacillary form of leprosy. Together, these data indicate that M. leprae induces a cell fate program which includes NUPR1 as part of the host response in the progressive form of leprosy.

BACKGROUNDReversal reactions (RRs) in leprosy are acute immune episodes marked by inflammation and bacterial clearance, offering a model to study the dynamics of host responses to Mycobacterium leprae. These episodes are often severe and difficult to treat, frequently progressing to permanent disabilities. We aimed to characterize the immune mechanisms and identify antimicrobial effectors during RRs.METHODSWe performed RNA-Seq on paired skin biopsy specimens collected from 9 patients with leprosy before and at RR diagnosis, followed by differential gene expression and functional analysis. A machine-learning classifier was applied to predict membrane-permeabilizing proteins. Antimicrobial activity was assessed in M. leprae-infected macrophages and axenic cultures.RESULTSIn the paired pre-RR and RR biopsy specimens, a 64-gene antimicrobial response signature was upregulated during RR and correlated with reduced M. leprae burden. Predicted upstream regulators included IL-1β, TNF, IFN-γ, and IL-17, indicating activation of both the Th1 and Th17 pathways. A machine-learning classifier identified 28 genes with predicted membrane-permeabilizing antimicrobial activity, including S100A8. Four proteins (S100A7, S100A8, CCL17, and CCL19) demonstrated antimicrobial activity against M. leprae in vitro. Scanning electron microscopy revealed membrane damage in bacteria exposed to these proteins.CONCLUSIONRR is associated with a robust antimicrobial gene program regulated by Th1 and Th17 cytokines. We identified potentially novel host antimicrobial effectors that showed activity against M. leprae, suggesting potential strategies to bolster Th1 and Th17 responses for combating intracellular mycobacterial infections.FUNDINGNIH grants R01 AI022553, R01 AR040312, R01 AR073252, R01 AI166313, R01 AI169526, P50 AR080594, and 4R37 AI052453-21 and National Science Foundation (NSF) grant DMR2325840.

The COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified. Given that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1β) on the IFN-g-induced NO production and its antiviral efficacy. Treatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1β enhanced IFN-γ induction of NO, but it had little effect on antiviral activity. Given that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.

[T.sub.H]17 cell subpopulations have been defined that contribute to inflammation and homeostasis, yet the characteristics of [T.sub.H]17 cells that contribute to host defense against infection are not clear. To elucidate the antimicrobial machinery of the [T.sub.H]17 subset, we studied the response to Cutibacterium acnes, a skin commensal that is resistant to IL-26, the only known [T.sub.H]17-secreted protein with direct antimicrobial activity. We generated C. acnes-specific antimicrobial [T.sub.H]17 clones ([sub.AM][T.sub.H]17) with varying antimicrobial activity against C. acnes, which we correlated by RNA sequencing to the expression of transcripts encoding proteins that contribute to antimicrobial activity. Additionally, we validated that [sub.AM][T.sub.H]17- mediated killing of C. acnes and bacterial pathogens was dependent on the secretion of granulysin, granzyme B, perforin, and histone H2B. We found that am[T.sub.H]17 cells can release fibrous structures composed of DNA decorated with histone H2B that entangle C. acnes that we call T cell extracellular traps (TETs). Within acne lesions, H2B and IL-17 colocalized in CD[4.sup.+] T cells, in proximity to TETs in the extracellular space composed of DNA decorated with H2B. This study identifies a functionally distinct subpopulation of [T.sub.H]17 cells with an ability to form TETs containing secreted antimicrobial proteins that capture and kill bacteria.

Abstract Granulomas are complex cellular structures comprised predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated single cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RR), a dynamic process in which some patients with disseminated lepromatous leprosy (L-lep) transition towards self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions, and regulated by IFN-γ and IL-1β. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts contribute to the antimicrobial response. Competing Interest Statement A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Cogen Therapeutics, Orche Bio, and Dahlia Biosciences.

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type I IFNs and antibody production. We performed dual RNAseq on patient lesions, identifying a continuum of distinct bacterial states that are linked to the host immune response. The bacterial burden, represented by the fraction of bacterial transcripts, correlates with a host type I IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial transcriptional activity, defined by the bacterial mRNA/rRNA ratio, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

Understanding mechanisms of pathogenesis and protection in human tuberculosis (TB) remain major global health challenges. While organized granulomas have long been the focus of TB research, growing evidence for asymptomatic transmission highlights the need to study earlier disease stages, particularly TB pneumonia, which remains underexplored. Defining the alveolar immune niche that governs bacillary expansion before granuloma formation is essential for interrupting transmission. Here, we integrate spatial transcriptomics, single cell RNA sequencing, and high resolution imaging of human lung biopsies to map early TB pneumonia and compare with adjacent granulomas within the same tissues. Pneumonic alveolar spaces were dominated by TREM2-associated macrophages, characterized by sparse T cell infiltration, minimal antimicrobial gene expression, and abundant antigens and transcripts. In contrast, granuloma cores were enriched for antimicrobial pathways, were surrounded by multiple cell types that walled off infection, and contained comparatively fewer bacterial markers. Our findings identify TREM2 positive 'foamy' macrophages as a key permissive alveolar niche for survival and growth. These cells represent an attractive target for early intervention to restrict infection and limit transmission. Early TB pneumonia defines an alveolar niche that fosters bacterial persistence and transmission before granuloma formation.

CoronaVac, an inactivated SARS-CoV-2 vaccine, has been approved for emergency use in several countries. However, its immunogenicity in immunocompromised individuals has not been well established. We initiated a prospective phase 4 controlled trial (no. NCT04754698, CoronavRheum) in 910 adults with autoimmune rheumatic diseases (ARD) and 182 age- and sex-frequency-matched healthy adults (control group, CG), who received two doses of CoronaVac. The primary outcomes were reduction of ≥15% in both anti-SARS-CoV-2 IgG seroconversion (SC) and neutralizing antibody (NAb) positivity 6 weeks (day 69 (D69)) after the second dose in the ARD group compared with that in the CG. Secondary outcomes were IgG SC and NAb positivity at D28, IgG titers and neutralizing activity at D28 and D69 and vaccine safety. Prespecified endpoints were met, with lower anti-SARS-Cov-2 IgG SC (70.4 versus 95.5%, P  < 0.001) and NAb positivity (56.3 versus 79.3%, P  < 0.001) at D69 in the ARD group than in the CG. Moreover, IgG titers (12.1 versus 29.7, P  < 0.001) and median neutralization activity (58.7 versus 64.5%, P  = 0.013) were also lower at D69 in patients with ARD. At D28, patients with ARD presented with lower IgG frequency (18.7 versus 34.6%, P  < 0.001) and NAb positivity (20.6 versus 36.3%, P  < 0.001) than that of the CG. There were no moderate/severe adverse events. These data support the use of CoronaVac in patients with ARD, suggesting reduced but acceptable short-term immunogenicity. The trial is still ongoing to evaluate the long-term effectiveness/immunogenicity. In a large prospective phase 4 trial, vaccination with CoronaVac, an inactivated SARS-CoV-2 vaccine, elicited significantly lower virus-specific IgG antibodies and neutralizing antibodies in patients with autoimmune rheumatic diseases than in age- and sex-matched healthy control trial participants.