The prevailing model of protective immunity to tuberculosis is that CD4 T cells produce the cytokine IFN-γ to activate bactericidal mechanisms in infected macrophages. Although IFN-γ-independent CD4 T cell based control of M. tuberculosis infection has been demonstrated in vivo it is unclear whether CD4 T cells are capable of directly activating macrophages to control infection in the absence of IFN-γ. We developed a co-culture model using CD4 T cells isolated from the lungs of infected mice and M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs) to investigate mechanisms of CD4 dependent control of infection. We found that even in the absence of IFN-γ signaling, CD4 T cells drive macrophage activation, M1 polarization, and control of infection. This IFN-γ-independent control of infection requires activation of the transcription factor HIF-1α and a shift to aerobic glycolysis in infected macrophages. While HIF-1α activation following IFN-γ stimulation requires nitric oxide, HIF-1α-mediated control in the absence of IFN-γ is nitric oxide-independent, indicating that distinct pathways can activate HIF-1α during infection. We show that CD4 T cell-derived GM-CSF is required for IFN-γ-independent control in BMDMs, but that recombinant GM-CSF is insufficient to control infection in BMDMs or alveolar macrophages and does not rescue the absence of control by GM-CSF-deficient T cells. In contrast, recombinant GM-CSF controls infection in peritoneal macrophages, induces lipid droplet biogenesis, and also requires HIF-1α for control. These results advance our understanding of CD4 T cell-mediated immunity to M. tuberculosis, reveal important differences in immune activation of distinct macrophage types, and outline a novel mechanism for the activation of HIF-1α. We establish a previously unknown functional link between GM-CSF and HIF-1α and provide evidence that CD4 T cell-derived GM-CSF is a potent bactericidal effector.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFa, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1,-11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell, Roncaioli et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides protection from Shigella colonization of the intestinal epithelium, but only in the absence of both NAIP-NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

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Diagnosis of pulmonary tuberculosis (PTB) in context of HIV infection remains challenging due 25 to the paucibacillary nature of the disease. Mycobacterium tuberculosis thymidylate kinase 26 (TMKmt) has been validated as a novel biomarker with high detection limits for PTB in sputum. 27 We aimed to clinically test TMKmt as a diagnostic biomarker for pulmonary tuberculosis among 28 HIV positive individuals at Makerere University Joint AIDS Program Immuno Suppression 29 Syndrome clinic. 30 Methods 31 A total of 120 participants with presumptive PTB were enrolled in a cross- sectional study 32 between January and September 2018. Venous blood and expectorated spot sputum was obtained 33 from 116 consenting participants. Tuberculosis culture was performed on sputum as the gold 34 standard to confirm PTB status while direct ELISAs were performed on sputum and serum to 35 determine the level of TMKmt antigen. Sensitivity, specificity and receiver operator 36 characteristic curves were used to assess the precision of TMKmt in diagnosing PTB. 37 Results 38 Of the 116 participants, only 22 (19%) were PTB positive based on either Lowenstein Jensen or 39 Mycobacterial growth inhibition tube `culture. The mean sputum TMKmt levels were 40 significantly higher in PTB positive individuals (4.889 ± 0.135) ng/ml as compared to PTB 41 negative individuals (4.303 ± 0.07295) ng/ml (t-test, P<0.0005). At a cut off value of > 4.683 42 ng/ml, the test had a sensitivity of 73% (95% CI, 49.78% - 89.27%) and a specificity of 72% 43 (95% CI, 62.15% - 81.07%). These results were supported by a receiver operator analysis which 44 showed an area under the curve of 0.75 (95% CI, 0.63 – O.86). 3 45 Conclusion 46 Sputum TMKmt is a potential diagnostic biomarker for active pulmonary tuberculosis. This 47 forms an alternate test that would replace smear microscopy and overcome the current TB 48 diagnostic challenges.